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.
|Advisor:||Xiao, Xiao, Huang, Leaf|
|Commitee:||Asokan, Aravind, Hingtgen, Shawn, Liu, Rihe|
|School:||The University of North Carolina at Chapel Hill|
|School Location:||United States -- North Carolina|
|Source:||DAI-B 79/10(E), Dissertation Abstracts International|
|Subjects:||Bioengineering, Molecular biology, Virology|
|Keywords:||Adeno-associated virus, Gene therapy, Large-scale production, Packaging cell line, Producer cell line, Vector production|
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