Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery

Abstract

Adeno-associated virus (AAV) is being used successfully in gene therapy. Different serotypes of AAV target specific organs and tissues with high efficiency. There exists an increasing demand to manufacture various AAV serotypes in large quantities for pre-clinical and clinical trials. A generic and scalable method has been described in this study to efficiently produce AAV serotypes (AAV1-9) by transfection of a fully characterized cGMP HEK293SF cell line grown in suspension and serum-free medium. First, the production parameters were evaluated using AAV2 as a model serotype. Second, all nine AAV serotypes were produced successfully with yields of 10(13)Vg/L cell culture. Subsequently, AAV2 and AAV6 serotypes were produced in 3-L controlled bioreactors where productions yielded up to 10(13)Vg/L similar to the yields obtained in shake-flasks. For example, for AAV2 10(13)Vg/L cell culture (6.8×10(11)IVP/L) were measured between 48 and 64h post transfection (hpt). During this period, the average cell specific AAV2 yields of 6800Vg per cell and 460IVP per cell were obtained with a Vg to IVP ratio of less than 20. Successful operations in bioreactors demonstrated the potential for scale-up and industrialization of this generic process for manufacturing AAV serotypes efficiently.

Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies

“…Major advances have occurred in the development of Adeno-associated viruses (AAVs) as gene delivery vectors over the last two decades, including improvements in large scale vector production to support clinical trials (Chahal et al 2014; Martin et al 2013; Mietzsch et al 2014). Significantly, recent successes in clinical trials worldwide have resulted in the approval of the use of the first AAV gene therapy product in Europe for the treatment of lipoprotein lipase deficiency (Pollack 2012), and numerous clinical trials are in progress for many other disease targets (Bainbridge et al 2008; Brantly et al 2009; Daniel Gaudet 2012; Ginn et al 2013; Maguire et al 2009; Maguire et al 2008; Mendell et al 2009; Smith et al 2013; Wierzbicki et al 2013).…”

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.…”

Rational plasmid design and bioprocess optimization to enhance recombinant adeno‐associated virus (AAV) productivity in mammalian cells

“…Not only a further increase in rAAV titer is expected but cost of goods will be significantly reduced by such an approach. In a further experiment, performance of anchorage‐ and serum‐dependent HEK293T cells was tested against well‐established HEK293SF‐3F6 suspension cells cultivated in a chemically defined medium . With regard to regulatory concerns when using animal‐derived components, serum‐free cultivation is a highly desirable goal for vector production .…”

“…Productions of AAV vectors in 125 mL shake flasks (Corning Incorporation, Corning, NY, USA) were performed using suspension HEK293SF‐3F6 cells. Cultivation, transfection, harvest (72 h post transfection) and purification approaches were performed as described previously .…”

“…Clinical success of rAAV‐based gene therapy together with rather low transduction efficiency in vivo raised the demand for rAAV vectors . Economical large‐scale rAAV manufacturing is still hampered by largely inefficient or non‐robust production processes .…”