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Cell line and process development strategies to establish HEK293-based recombinant adeno-associated virus production platforms
Date
2024
Authors
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Publisher
University of Delaware
Abstract
Recombinant adeno associated virus (rAAV) vectors have become popular delivery vehicles for in vivo gene therapies, but demand for rAAVs continues to outpace supply due to low process yields and manufacturing capacity limitations. Transient transfection of human embryonic kidney 293 (HEK293) cells is the current gold standard rAAV manufacturing process, but this system is not readily scalable to produce vector for large patient populations. Stable producer cell lines (PCLs) can be cultured at much larger scales than transient processes, making stable platforms attractive for manufacturing of AAVs, particularly those with higher demand or dosing requirements. However, generation of rAAV PCLs is difficult because sequences encoding AAV replication (Rep) genes, AAV capsid (Cap) genes, and helper adenovirus (AdV) genes must be integrated into the host cell under the control of inducible promoters. ☐ In this work, we established protein production, analytical characterization, and cell line development workflows to support the design and screening of inducible gene constructs that can be used for the generation of HEK293-derived rAAV PCLs. Initial studies focused on establishing well plate scale transient transfection and PCL development workflows using the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (rRBD) as a model protein. The top four transient expression process and stable clones identified in the screens were scaled up for culture in shake flasks, reaching titers up to 100 mg/L and 140 mg/L, respectively by ELISA. ☐ Subsequent work focused on establishing traditional, triple transient transfection platforms for rAAV production in HEK293 leveraging the resources of the Advanced Mammalian Biomanufacturing Innovation Center (AMBIC). The plasmids for these systems encode the necessary rAAV production genes across three constructs (pRepCap, pHelper, pGenome) containing constitutively expressed, wild type viral gene sequences. A commercial rAAV cell line and culture medium was first used to establish workflows for rAAV production and methods to measure vector genome (VG) and capsid particle (CP) titers. A model-based DOE approach was then used to optimize plasmid DNA and transfection reagent complexation parameters for the commercial system. To develop an in-house AMBIC rAAV production platform, a chemically defined cell culture medium was developed, and the previously utilized DOE approaches were employed to refine process parameters. Work to develop both the commercial and AMBIC systems enabled us to define operating ranges for complexation and plasmid ratio setpoints, and set benchmarks for expected titer ranges. ☐ We then developed recombinase mediated cassette exchange (RMCE) and PiggyBac (PB) transposase mediated stable gene insertion workflows for the design and testing of inducible gene cassettes expressing fluorescent reporter and rAAV production genes. The RMCE system was used to screen doxycycline (dox) inducible cassette configurations expressing a fluorescent reporter, and the cassette with the highest fold inducibility and lowest level of leak was used to systematically compare the cytotoxicity of stable rAAV production gene expression. Stable dox induction of two production genes, AAV replication gene Rep78 and adenovirus helper gene E4-34K, suppressed cell growth and caused viability to decline over the three days in culture. This result motivated the rapid screening of rAAV production gene cassettes designed to modulate cytotoxic protein expression using a transient inducible expression system. The maximum VG and CP titers reached in our transient inducible study were 4.6×10E10 VG/mL and 1.4×10E12 CP/mL, which were 49% and 74% of those from cultures transfected with wild type sequences, respectively. The hyperactive PB system was then applied to facilitate transposition of rAAV-eGFP genome sequences to support production of >10E9 secreted vector genomes per mL of culture and of the rAAV Cap8 to enable synthesis of >10E12 secreted total capsids per mL of culture. These results demonstrate the potential of the RMCE and hyPB systems to be applied to develop strategies for the generation of full rAAV producer cell lines.
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Keywords
Biotechnology, Cell line development, HEK293, Upstream process development