Generation and Characterization of Adeno-Associated Virus Producer Cell Lines for Research and Preclinical Vector Production
| Autor: | Yuxia Luo, Eric Pastor, Bruce Sol, John M. Martin, Karen A. Vincent, Michelle Joubert, Amy Frederick, Samuel C. Wadsworth, Donna Armentano, Robert B. Jackson, Francis Poulin |
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| Rok vydání: | 2013 |
| Předmět: |
viruses
Genetic Vectors Population Biology Transfection medicine.disease_cause Applied Microbiology and Biotechnology Virus Viral Proteins Plasmid Genetics medicine Humans Vector (molecular biology) education Adeno-associated virus Genetics (clinical) Selectable marker Pharmacology education.field_of_study Virus Assembly Genetic Therapy Dependovirus Virology Molecular biology Recombinant Proteins Cell culture Molecular Medicine HeLa Cells |
| Zdroj: | Human Gene Therapy Methods. 24:253-269 |
| ISSN: | 1946-6544 1946-6536 |
| DOI: | 10.1089/hgtb.2013.046 |
| Popis: | Adeno-associated virus (AAV) producer cell lines represent an effective method for large-scale production of AAV vectors. We set out to evaluate and characterize the use of an abbreviated protocol to generate "masterwells" (MWs; a nonclonal cell population) as a platform for research and preclinical vector production. In this system, a single plasmid containing three components, the vector sequence, the AAV rep, and cap genes, and a selectable marker gene is stably transfected into HeLaS3 cells. Producer cell lines generating an AAV2 vector expressing a secreted form of human placental alkaline phosphatase (SEAP) have been created. Several MWs showed vector yields in the 5×10(4) to 2×10(5) DNase-resistant particles/cell range, and the productivity was stable over60 population doublings. Integrated plasmid copy number in three high-producing MWs ranged from approximately 12 to 50; copies were arranged in a head-to-tail configuration. Upon infection with adenovirus, rep/cap copy number was amplified approximately 100-fold and high yield appeared to be dependent on the extent of amplification. Rep/cap gene expression and vector packaging both reached a peak at 48 hr postinfection. AAV2-SEAP vector was produced in 1-liter shaker culture and purified for assessment of vector quality and potency. The data showed that the majority of the capsids from the MWs contained vector DNA (≥70%) and that purified vector was free of replication-competent AAV. In vitro and in vivo analyses demonstrated that potency of the producer cell-derived vector was comparable to vector generated via the standard transfection method. |
| Databáze: | OpenAIRE |
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