Standardized, Scalable, and Timely Flexible Adeno-Associated Virus Vector Production Using Frozen High-Density HEK-293 Cell Stocks and CELLdiscs

Abstract

Adeno-associated virus (AAV) vectors currently represent the most attractive platform for viral gene therapy and are also valuable research tools to study gene function or establish disease models. Consequently, many academic labs, core facilities, and biotech/pharma companies meanwhile produce AAVs for research and early clinical development. Whereas fast, universal protocols for vector purification (downstream processing) are available, AAV production using adherent HEK-293 cells still requires time-consuming passaging and extensive culture expansion before transfection. Moreover, most scalable culture platforms require special equipment or extensive method development. To tackle these limitations in upstream processing, this study evaluated frozen high-density cell stocks as a ready-to-seed source of producer cells, and further investigated the multilayered CELLdisc culture system for upscaling. The results demonstrate equal AAV productivity using frozen cell stock–derived cultures compared to conventionally cultured cells, as well as scalability using CELLdiscs. Thus, by directly seeding freshly thawed cells into CELLdiscs, AAV production can be easily upscaled and efficiently standardized to low-passage, high-viability cells in a timely flexible manner, potentially dismissing time-consuming routine cell culture work. In conjunction with a further optimized iodixanol protocol, this process enabled supply to a large-animal study with two high-yield AAV2 capsid variant batches (0.6–1.2 × 1015 vector genomes) in as little as 4 weeks.

Large-scale microcarrier culture of HEK293T cells and Vero cells in single-use bioreactors

“…Commonly used gene transfer vehicles are viral vectors, which include lentivirus, adenovirus, adenovirus associated virus, retrovirus, etc. (Humphreys and Sebastian 2018; Strobel et al 2019). Currently, thousands of gene therapy projects and viral vaccine projects are in Investigation New Drug phases or clinical trial phases.…”

The Current and Future State of Vaccines, Antivirals and Gene Therapies Against Emerging Coronaviruses

“…AAV-based passive immunization could perfectly fill the vacuum by protecting the population before the arrival of a vaccine. Since AAV is a platform technology, anti-viral packages can be swapped and tested quickly, within a month (Strobel et al, 2019). After administration in animals, protection can be achieved in less than a week, faster than any vaccine strategies.…”

The biphasic and age-dependent impact of Klotho on hallmarks of aging and skeletal muscle function

“…Recombinant AAV8 was used as vector for expressing murine Klotho GlutaMAX-I + 10% fetal calf serum (Gibco/Thermo Fisher Scientific) and transfected as previously described (80). AAV purification via polyethylenglycol precipitation, iodixanol gradient, ultrafiltration and sterile filtering was conducted as described previously (79). Genomic titers of purified AAV8 vector stocks were determined by isolation of viral DNA and subsequent qPCR analysis using primers specific for the LP-1 promotor with the following primer sequences; forward: GACCCCCTAAAATGGGCAAA.…”