Gene therapy developers targeting diseases requiring high dose therapies face a challenge. Current vector production cell lines and genetic manipulation techniques are too costly for large-scale manufacturing, according to new research.

The study, by scientists at Portugal’s Instituto de Biologia Experimental e Tecnológica (iBET), looked at areas of gene therapy production where innovation is needed.

“The biggest challenges for rAAV-based gene therapies are bioprocess scalability and manufacturing costs,” says lead author and iBET CEO Paula Alves, PhD, citing the limited choice of stable producer cell lines for vectors as the major issue.

Most gene therapy production processes, including those used for Spark Therapeutics’ Luxturna (voretigene neparvovec), rely on transient transfection of HEK293 cells which, Alves notes, is only feasible at small scale.

“For Phase I and II clinical trials and for non-systemic therapies, such as the FDA-approved treatment for congenital blindness, where the therapeutic dosage is low, transient transfection is not so problematic. Although cost of goods still accounts for 20% of final production cost, the transient transfection scales are still feasible,” Alves tells GEN.

“However, for larger scales and commercial manufacturing, transient transfection is not adequate, citing a trial candidate Duchenne muscular dystrophy gene therapy for which the dose is higher, around 1014 vg/kg rAAV per patient-as an example.

“Implementing transfection protocols at large scale and the need for high amounts of GMP grade plasmids and costly transfection reagents are big challenges.”

Improved AAV production

One potential solution would be to improve the cell lines used to manufacture recombinant adeno-associated (rAAV) vectors explains Alves, who says “this will facilitate scalability, reduce costs, and accelerate regulatory compliance.

“There are some commercially available inducible systems, including CEVEC Pharmaceuticals’ platform, where rAAV production is triggered by the addition of tetracycline. Another example is the HelaS3 based producer cell line system that is already used by a number of companies.

“However, these systems still need to be improved because the titers are low. At iBET, we have several projects specially focusing on cell line development that, combined with integrated continuous bioprocessing approaches, will hopefully lead to better rAAV production processes using stable cell lines.”

In their study, Alves and her team also say the lack of defined critical quality attributes for rAAV products holds back the “development of robust production methods.” Solving the issue will require investment in technology.

“From my point of view, this is not because this is an immature field, but rather because there aren’t proper analytical tools for monitoring rAAV production,” she continues. “At iBET we believe that better process understanding will be needed to attain high quality rAAVs, and we need analytics to do so. Thus, our strategy is to build teams with upstream, downstream and analytics scientists.

“Only with this multidisciplinary approach will the development of suitable bioanalytics for the analysis of in-process rAAV samples be possible.”