ingest cdrApp 2018-06-13T19:47:49.024Z 51cd2fe2-3fd7-401f-a923-a97bc3db68a2 modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-06-13T21:08:21.516Z Setting exclusive relation addDatastream MD_TECHNICAL fedoraAdmin 2018-06-13T21:08:32.839Z Adding technical metadata derived by FITS addDatastream MD_FULL_TEXT fedoraAdmin 2018-06-13T21:08:56.659Z Adding full text metadata extracted by Apache Tika modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-06-13T21:09:19.182Z Setting exclusive relation modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-07-11T09:56:03.799Z Setting invalid vocabulary terms modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-07-11T09:56:15.926Z modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-07-16T18:19:16.467Z Setting invalid vocabulary terms modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-07-18T05:58:41.728Z Setting invalid vocabulary terms modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-08-16T19:05:57.825Z Setting invalid vocabulary terms modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-08-16T19:06:09.185Z modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-09-27T14:50:26.242Z Setting invalid vocabulary terms modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-09-27T14:50:37.510Z modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-10-12T06:03:40.815Z Setting invalid vocabulary terms modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-10-12T06:03:52.264Z modifyDatastreamByValue RELS-EXT fedoraAdmin 2019-03-21T15:51:04.260Z Setting invalid vocabulary terms modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2019-03-21T15:51:16.296Z Tejash Patel Author Pharmaceutical Sciences Process Design for Scalable Recombinant Adeno-Associated Virus The versatility of recombinant adeno-associated viruses has garnered significant attention from investors, pharmaceutical companies, and regulatory agencies as more therapies using this vector are finding success in preclinical and late-stage clinical trials, even some reaching the market approval stage in the US. A number of vector production methods have been developed to generate the necessary clinical grade vectors that have high potency, and have high-titers to reduce general manufacturing and therapeutic costs. However, each of these production methods inherently have their own drawbacks, whether it is the concomitantly produced helper vectors used to make the AAV or the quality of the AAV itself. We previously developed a HEK293-cell based producer cell line method for high titer and high potency rAAV vectors. However, this method requires significant time-intensive generation of novel producer cell lines for every rAAV vector of interest and furthermore the E1-, E3-deleted adenovirus is only occupied at the E1-region, leaving the space available in the E3-region unused. In this body of work, we discuss a novel adenovirus construct that utilizes a packaging cell line to generate a high titer and high quality rAAV, and attempts to remove the accompanying adenovirus in the final product batch. The improvements made in this system are: 1) the development of a one-step cloning of a rAAV vector cassette into the E1-region of the adenovirus, 2) use of an efficiently generated packaging cell line to universally package the rAAV vector provided in trans by the adenovirus, 3) high vector yields on different rAAV inverted terminal repeat designs provided by the adenovirus, and 4) high quality, low. empty-particle containing rAAV product. We attempt to remove contaminating entities in the rAAV production method, using a selective precipitating agent called domiphen bromide. Although this detergent is efficient in removing contaminating materials such as DNA and adenovirus, there are major difficulties to reduce interactions with certain serotypes of AAV. Therefore, an alternative method for removing adenovirus is necessary, possibly by high hydrostatic pressure or nanofilters. The simple adenovirus construct coupled with the packaging cell line can be a pivotal method for large scale AAV vector production. Winter 2018 2018 Bioengineering Molecular biology Virology Adeno-Associated Virus, Gene Therapy, Large-Scale Production, Packaging Cell Line, Producer Cell Line, Vector Production eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Pharmaceutical Sciences Xiao Xiao Thesis advisor Leaf Huang Thesis advisor Shawn Hingtgen Thesis advisor Aravind Asokan Thesis advisor Rihe Liu Thesis advisor text Tejash Patel Author Pharmaceutical Sciences Process Design for Scalable Recombinant Adeno-Associated Virus The versatility of recombinant adeno-associated viruses has garnered significant attention from investors, pharmaceutical companies, and regulatory agencies as more therapies using this vector are finding success in preclinical and late-stage clinical trials, even some reaching the market approval stage in the US. A number of vector production methods have been developed to generate the necessary clinical grade vectors that have high potency, and have high-titers to reduce general manufacturing and therapeutic costs. However, each of these production methods inherently have their own drawbacks, whether it is the concomitantly produced helper vectors used to make the AAV or the quality of the AAV itself. We previously developed a HEK293-cell based producer cell line method for high titer and high potency rAAV vectors. However, this method requires significant time-intensive generation of novel producer cell lines for every rAAV vector of interest and furthermore the E1-, E3-deleted adenovirus is only occupied at the E1-region, leaving the space available in the E3-region unused. In this body of work, we discuss a novel adenovirus construct that utilizes a packaging cell line to generate a high titer and high quality rAAV, and attempts to remove the accompanying adenovirus in the final product batch. The improvements made in this system are: 1) the development of a one-step cloning of a rAAV vector cassette into the E1-region of the adenovirus, 2) use of an efficiently generated packaging cell line to universally package the rAAV vector provided in trans by the adenovirus, 3) high vector yields on different rAAV inverted terminal repeat designs provided by the adenovirus, and 4) high quality, low. empty-particle containing rAAV product. We attempt to remove contaminating entities in the rAAV production method, using a selective precipitating agent called domiphen bromide. Although this detergent is efficient in removing contaminating materials such as DNA and adenovirus, there are major difficulties to reduce interactions with certain serotypes of AAV. Therefore, an alternative method for removing adenovirus is necessary, possibly by high hydrostatic pressure or nanofilters. The simple adenovirus construct coupled with the packaging cell line can be a pivotal method for large scale AAV vector production. Winter 2018 2018 Bioengineering Molecular biology Virology Adeno-Associated Virus, Gene Therapy, Large-Scale Production, Packaging Cell Line, Producer Cell Line, Vector Production eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Pharmaceutical Sciences Xiao Xiao Thesis advisor Leaf Huang Thesis advisor Shawn Hingtgen Thesis advisor Aravind Asokan Thesis advisor Rihe Liu Thesis advisor text Tejash Patel Author Pharmaceutical Sciences Process Design for Scalable Recombinant Adeno-Associated Virus The versatility of recombinant adeno-associated viruses has garnered significant attention from investors, pharmaceutical companies, and regulatory agencies as more therapies using this vector are finding success in preclinical and late-stage clinical trials, even some reaching the market approval stage in the US. A number of vector production methods have been developed to generate the necessary clinical grade vectors that have high potency, and have high-titers to reduce general manufacturing and therapeutic costs. However, each of these production methods inherently have their own drawbacks, whether it is the concomitantly produced helper vectors used to make the AAV or the quality of the AAV itself. We previously developed a HEK293-cell based producer cell line method for high titer and high potency rAAV vectors. However, this method requires significant time-intensive generation of novel producer cell lines for every rAAV vector of interest and furthermore the E1-, E3-deleted adenovirus is only occupied at the E1-region, leaving the space available in the E3-region unused. In this body of work, we discuss a novel adenovirus construct that utilizes a packaging cell line to generate a high titer and high quality rAAV, and attempts to remove the accompanying adenovirus in the final product batch. The improvements made in this system are: 1) the development of a one-step cloning of a rAAV vector cassette into the E1-region of the adenovirus, 2) use of an efficiently generated packaging cell line to universally package the rAAV vector provided in trans by the adenovirus, 3) high vector yields on different rAAV inverted terminal repeat designs provided by the adenovirus, and 4) high quality, low. empty-particle containing rAAV product. We attempt to remove contaminating entities in the rAAV production method, using a selective precipitating agent called domiphen bromide. Although this detergent is efficient in removing contaminating materials such as DNA and adenovirus, there are major difficulties to reduce interactions with certain serotypes of AAV. Therefore, an alternative method for removing adenovirus is necessary, possibly by high hydrostatic pressure or nanofilters. The simple adenovirus construct coupled with the packaging cell line can be a pivotal method for large scale AAV vector production. Winter 2018 2018 Bioengineering Molecular biology Virology Adeno-Associated Virus, Gene Therapy, Large-Scale Production, Packaging Cell Line, Producer Cell Line, Vector Production eng Doctor of Philosophy Dissertation Pharmaceutical Sciences Xiao Xiao Thesis advisor Leaf Huang Thesis advisor Shawn Hingtgen Thesis advisor Aravind Asokan Thesis advisor Rihe Liu Thesis advisor text University of North Carolina at Chapel Hill Degree granting institution Tejash Patel Creator Pharmaceutical Sciences Process Design for Scalable Recombinant Adeno-Associated Virus The versatility of recombinant adeno-associated viruses has garnered significant attention from investors, pharmaceutical companies, and regulatory agencies as more therapies using this vector are finding success in preclinical and late-stage clinical trials, even some reaching the market approval stage in the US. A number of vector production methods have been developed to generate the necessary clinical grade vectors that have high potency, and have high-titers to reduce general manufacturing and therapeutic costs. However, each of these production methods inherently have their own drawbacks, whether it is the concomitantly produced helper vectors used to make the AAV or the quality of the AAV itself. We previously developed a HEK293-cell based producer cell line method for high titer and high potency rAAV vectors. However, this method requires significant time-intensive generation of novel producer cell lines for every rAAV vector of interest and furthermore the E1-, E3-deleted adenovirus is only occupied at the E1-region, leaving the space available in the E3-region unused. In this body of work, we discuss a novel adenovirus construct that utilizes a packaging cell line to generate a high titer and high quality rAAV, and attempts to remove the accompanying adenovirus in the final product batch. The improvements made in this system are: 1) the development of a one-step cloning of a rAAV vector cassette into the E1-region of the adenovirus, 2) use of an efficiently generated packaging cell line to universally package the rAAV vector provided in trans by the adenovirus, 3) high vector yields on different rAAV inverted terminal repeat designs provided by the adenovirus, and 4) high quality, low. empty-particle containing rAAV product. We attempt to remove contaminating entities in the rAAV production method, using a selective precipitating agent called domiphen bromide. Although this detergent is efficient in removing contaminating materials such as DNA and adenovirus, there are major difficulties to reduce interactions with certain serotypes of AAV. Therefore, an alternative method for removing adenovirus is necessary, possibly by high hydrostatic pressure or nanofilters. The simple adenovirus construct coupled with the packaging cell line can be a pivotal method for large scale AAV vector production. Bioengineering Molecular biology Virology Adeno-Associated Virus; Gene Therapy; Large-Scale Production; Packaging Cell Line; Producer Cell Line; Vector Production eng Doctor of Philosophy Dissertation Pharmaceutical Sciences Xiao Xiao Thesis advisor Leaf Huang Thesis advisor Shawn Hingtgen Thesis advisor Aravind Asokan Thesis advisor Rihe Liu Thesis advisor text University of North Carolina at Chapel Hill Degree granting institution 2018 2018-12 Tejash Patel Author Pharmaceutical Sciences Process Design for Scalable Recombinant Adeno-Associated Virus The versatility of recombinant adeno-associated viruses has garnered significant attention from investors, pharmaceutical companies, and regulatory agencies as more therapies using this vector are finding success in preclinical and late-stage clinical trials, even some reaching the market approval stage in the US. A number of vector production methods have been developed to generate the necessary clinical grade vectors that have high potency, and have high-titers to reduce general manufacturing and therapeutic costs. However, each of these production methods inherently have their own drawbacks, whether it is the concomitantly produced helper vectors used to make the AAV or the quality of the AAV itself. We previously developed a HEK293-cell based producer cell line method for high titer and high potency rAAV vectors. However, this method requires significant time-intensive generation of novel producer cell lines for every rAAV vector of interest and furthermore the E1-, E3-deleted adenovirus is only occupied at the E1-region, leaving the space available in the E3-region unused. In this body of work, we discuss a novel adenovirus construct that utilizes a packaging cell line to generate a high titer and high quality rAAV, and attempts to remove the accompanying adenovirus in the final product batch. The improvements made in this system are: 1) the development of a one-step cloning of a rAAV vector cassette into the E1-region of the adenovirus, 2) use of an efficiently generated packaging cell line to universally package the rAAV vector provided in trans by the adenovirus, 3) high vector yields on different rAAV inverted terminal repeat designs provided by the adenovirus, and 4) high quality, low. empty-particle containing rAAV product. We attempt to remove contaminating entities in the rAAV production method, using a selective precipitating agent called domiphen bromide. Although this detergent is efficient in removing contaminating materials such as DNA and adenovirus, there are major difficulties to reduce interactions with certain serotypes of AAV. Therefore, an alternative method for removing adenovirus is necessary, possibly by high hydrostatic pressure or nanofilters. The simple adenovirus construct coupled with the packaging cell line can be a pivotal method for large scale AAV vector production. Winter 2018 2018 Bioengineering Molecular biology Virology Adeno-Associated Virus, Gene Therapy, Large-Scale Production, Packaging Cell Line, Producer Cell Line, Vector Production eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Pharmaceutical Sciences Xiao Xiao Thesis advisor Leaf Huang Thesis advisor Shawn Hingtgen Thesis advisor Aravind Asokan Thesis advisor Rihe Liu Thesis advisor text Tejash Patel Creator Pharmaceutical Sciences Program The versatility of recombinant adeno-associated viruses has garnered significant attention from investors, pharmaceutical companies, and regulatory agencies as more therapies using this vector are finding success in preclinical and late-stage clinical trials, even some reaching the market approval stage in the US. A number of vector production methods have been developed to generate the necessary clinical grade vectors that have high potency, and have high-titers to reduce general manufacturing and therapeutic costs. However, each of these production methods inherently have their own drawbacks, whether it is the concomitantly produced helper vectors used to make the AAV or the quality of the AAV itself. We previously developed a HEK293-cell based producer cell line method for high titer and high potency rAAV vectors. However, this method requires significant time-intensive generation of novel producer cell lines for every rAAV vector of interest and furthermore the E1-, E3-deleted adenovirus is only occupied at the E1-region, leaving the space available in the E3-region unused. In this body of work, we discuss a novel adenovirus construct that utilizes a packaging cell line to generate a high titer and high quality rAAV, and attempts to remove the accompanying adenovirus in the final product batch. The improvements made in this system are: 1) the development of a one-step cloning of a rAAV vector cassette into the E1-region of the adenovirus, 2) use of an efficiently generated packaging cell line to universally package the rAAV vector provided in trans by the adenovirus, 3) high vector yields on different rAAV inverted terminal repeat designs provided by the adenovirus, and 4) high quality, low. empty-particle containing rAAV product. We attempt to remove contaminating entities in the rAAV production method, using a selective precipitating agent called domiphen bromide. Although this detergent is efficient in removing contaminating materials such as DNA and adenovirus, there are major difficulties to reduce interactions with certain serotypes of AAV. Therefore, an alternative method for removing adenovirus is necessary, possibly by high hydrostatic pressure or nanofilters. The simple adenovirus construct coupled with the packaging cell line can be a pivotal method for large scale AAV vector production. 2018-12 2018 Bioengineering Molecular biology Virology Adeno-Associated Virus; Gene Therapy; Large-Scale Production; Packaging Cell Line; Producer Cell Line; Vector Production eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Xiao Xiao Thesis advisor Leaf Huang Thesis advisor Shawn Hingtgen Thesis advisor Aravind Asokan Thesis advisor Rihe Liu Thesis advisor text Patel_unc_0153D_17485.pdf uuid:fae582d1-ed79-4d74-a043-59d3c2907076 2020-06-13T00:00:00 proquest 2018-01-24T20:45:26Z application/pdf 83519278 affiliation|Pharmaceutical Sciences Program