Generation and Characterization of Adeno-Associated Virus Producer Cell Lines for Research and Preclinical Vector Production

Abstract

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 over >60 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.

OneBac 2.0:Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA

“…22 Recently, an AAV production system based on stable HeLa cell lines was presented that harbors the rep and cap genes and the ITR-flanked rAAV genome. 23 Considerable collateral packaging of rep and cap was reported, up to 0.62% and 0.25%, respectively, of vector genome-containing capsids. In rAAV2 vector lots for a clinical trial, the reported percentage of packaged cap genes ranged between 0.016% and 0.021%.…”

High-Density Recombinant Adeno-Associated Viral Particles are Competent Vectors forIn VivoTransduction

“…7 Encouraging preliminary results are moving rAAV gene therapy closer to a routine clinical application. Although there have been continuous and significant progressions toward flexible and scalable rAAV production in recent years, [8][9][10][11][12][13] a high demand still exists for more efficient vector production. This is partially due to the overwhelming amount of quality vectors needed to support preclinical research using large animal models and clinical trials.…”

Characteristics of Minimally Oversized Adeno-Associated Virus Vectors Encoding Human Factor VIII Generated Using Producer Cell Lines and Triple Transfection

“…18,19 However, a disadvantage of this process is the time needed to generate and characterize producer cell lines (PCLs), which can be shortened using non-clonal ''masterwells'' (MWs). 18,26 This study generated and characterized MWs producing AAVrh8R serotype vectors harboring 5.1 and 5.4 kb genomes encoding human FVIII (hFVIII). The data demonstrate the feasibility of the PCL method to produce slightly oversized rAAV vectors.…”

“…The AAV2 cap in pAF-SEAP-AAV2 26 was changed to AAVrh8R to gener-ate pTP-SEAP-caprh8R. The ITRs flanking the SEAP expression cassette were removed and replaced with a blunt-ended PvuI and SapI fragment containing the 5.1 kb FVIII vector genome from pITR-mTTR202-hFVIIIco-spA to generate pTP-FVIII-5.1-caprh8R.…”