Presented by: Jason King, business development manager, Oxgene, a WuXi Advanced Therapies company
Since joining the WuXi Advanced Therapies division of WuXi AppTec in March 2021, Oxgene has focused on helping gene therapy developers to optimize their viralvector production systems as they transition from preclinical and early clinical projects to good manufacturing practice (GMP)-grade manufacturing. King observed that early stages of gene therapy development nearly always involve systems for transient transfection rather than scalable platforms. Inducible producer cell lines, viral transduction systems, and other scalable solutions require firm establishment of genes of interest, expression promoters, and in some cases, vector serotypes. Thus, such systems leave little room for modifications. Alternatively, transient transfection platforms can leverage plasmids, human embryonic kidney (HEK) 293 cells, and other accessible and cost-effective options to facilitate small-scale studies, providing much needed flexibility during early stage optimization of therapeutic transgenes.
Kingâ€™s presentation described how his company developed its latest generation of transient transfection platforms. Oxgene recently optimized its three-plasmid expression construct for recombinant adenoassociated virus (rAAV) vectors. King noted that plasmids containing rAAV replicative (rep) and capsid (cap) genes have remained nearly unchanged for decades, following much the same structure as that found in wild-type AAV. Oxgene has reconfigured that plasmid, splitting the rep and cap frames apart and changing their arrangement. The cap gene now is expressed using a strong cytomegalovirus (CMV) promoter; the rep gene is expressed by an encephalomyocarditis virus (EMCV) promoter.
The reconfigured plasmid enables up to three-fold improvement in production of rAAV particles, even before downstream concentration steps. Although the plasmid had been developed and tested using AAV2, subsequent studies have shown that its benefits extend across all natural AAV serotypes. During internal assessment, the optimized platform generated average viral-genome titers of 1011 vg/mL for rAAV5, rAAV6, and rAAV8. Those results correspond respectively to three-, eight-, and six-fold increases in viral titers, compared with titers achieved using a standard repâ€“cap plasmid. The reconfigured system produced rAAV9 titers of ~1010 vg/mL. Equally important, King pointed out, is that the new platform generates a higher ratio of full to empty/partial capsids than does a conventional transient system. Oxgene scientists attribute such increases in productivity to efficient genome encapsidation.
Oxgeneâ€™s partners have achieved similar or better results using the reconfigured plasmid. During production of rAAV5 in a HEK293 cell line, one customer generated as much as 2.1 Ã— 1011 vg/mL, which amounts to ~105 genome-containing units per cell. The customer also recorded full-particle rates between 57% and 72%, which is 3â€“5Ã— higher than what can be achieved using a standard repâ€“cap plasmid. Such increases in productivity, King explained, facilitate subsequent purification steps and enable recovery of more functional virus particles than can be produced and purified when starting with a conventional transient system.
Addressing Oxgeneâ€™s other offerings, King explained that his companyâ€™s latest platform for LV production features high viral titers and a strong biosafety profile. The platform transfects four plasmids into highly scalable, suspensioncultureâ€“ adapted HEK293 cells that lack large T antigens. By optimizing the cell-seeding density and the amount of genetic cargo delivered to cells, Oxgene scientists have increased average LV infectious titers to levels between 108 TU/mL and 2 Ã— 108 TU/mL.
King emphasized that Oxgene and WuXi Advanced Therapies have harmonized their operations in ways that can facilitate a gene therapy companyâ€™s advancement through the drug development pathway. Both companies now use the same viral vector production platforms. Thus, customers that work with Oxgene during early development stages can expect high continuity of processes and materials if they engage WuXi Advanced Therapies for clinical- and commercial-grade manufacturing. King added that Oxgene now can leverage parent company WuXi AppTecâ€™s state-of-the-art plasmid production facilities in China to accelerate its viral vector operations. Together, Oxgene and WuXi Advanced Therapies can help gene therapy companies to advance seamlessly from preclinical work using transient platforms to clinical and commercial manufacturing using scalable viral-vector solutions.