Gene Therapy Viral Vector Production Platforms

Gene therapy has seen significant development and success in recent decades. In this article, leading companies share the most updated information on viral vector production platforms, including transient transfection, mammalian stable cell lines, and baculovirus/Sf9. They provided tips on overcoming production challenges and accelerating gene therapy development.

Gene therapy, which introduces modified genetic material into a patient to treat disease, can be applied to replace, silence, add, or edit disease-causing genes. Since its emergence in the early 90s, it has been mainly developed to treat rare diseases—the first successful human gene therapy treated adenosine deaminase deficiency severe combined immunodeficiency disease (ADA-SCID) in a four-year-old girl.¹ However, it took almost 30 years until the first approved therapy, Kymriah^® (tisagenlecleucel), entered the market in 2017.²

Research in this area has witnessed significant development in the past 10 years due to advancements in different viral vector production platforms.³ After years of research, scientists started understanding the biological differences between viral vectors, becoming more armed to choose the best vector for different applications. "Different adeno-associated virus (AAV) serotypes can be used to target specific tissues and organs, whereas lentiviruses can hold larger genes and integrate into target cell genomes, making them ideal for ex vivo gene-modified cell therapies," explains Dirk Lange, Head of Life Science Services of MilliporeSigma.

According to Lange, "There is a better understanding of viral vectors and how they can treat different genetic diseases and cancers. We are starting to see therapies advance through clinical trials targeted toward larger patient populations and chronic conditions."

To date, the FDA has approved 13 therapies that utilize 5 different types of viral vectors: adenovirus (Ad vectors), adeno-associated virus (AAV), lentivirus, retroviral, and herpes simplex virus.^4,5

Viral vector production platforms

Viral vectors can be produced using transient transfection, mammalian stable cell lines, or infecting the baculovirus/Sf9 system. The choice of platform for viral vector production will depend on the final application. Transient transfection is better for short-term expression, including gene knockdown or silencing with inhibitory RNAs and protein production. Stable cell lines will be valuable for long-term pharmacology and genetic regulation studies.

Transient transfection

Some of the challenges in using cell lines can be overcome with transient transfections, which are fast and more flexible. In transient transfection, the cell will produce the gene of interest for a limited time, and until you start manufacturing, you can change the vector. Nevertheless, transient platforms are complex, challenging to scale, and generate more variability between batches due to plasmid contamination.

Mammalian stable cell lines

Mammalian producer cell lines, such as HEK-293, CHO, and Vero cells, are the most predictable choice for producing high-quality viral vectors. In stable cell lines, the genetic sequence and all the genes needed to make the viral particles are introduced into the cell using a virus, which is the most effective way to integrate the sequence in the cell genome.

Stable cell lines are simple and accessible platforms to scale up production with low batch variability. However, this platform lacks flexibility since you cannot change the sequence of interest or the vector type after production. Moreover, immortalized or transformed cell lines can generate genotoxicity and carry harmful impurities.

Baculovirus/Sf9 system

The baculovirus expression vector (BEV) system in Spodoptera frugiperda insect cells (Sf9) is an alternative method to mammalian cell lines. The Baculovirus/Sf9 is a flexible, clinical-grade, and high-titer large-scale vector production system with reduced encapsulation of contaminating DNAs. However, it generates viral vectors with diminished infectivity, primarily because of non-mammalian post-translational modifications, thus demanding higher doses for optimal efficacy.

How to overcome manufacturing challenges

Common challenges in viral vector manufacturing include choosing a suitable production system, refining downstream processing, and establishing standardized chemistry, manufacturing, and control procedures along with quality assays.⁶

Experts widely agree that viral vector manufacturing needs to be optimized and validated. Essential factors like selecting the appropriate cell line, optimizing culture conditions, refining transfection methods, and determining optimal harvest times should be finely tuned. This is crucial to ensure that the process achieves the best results in terms of yield, cost, quality, and safety.

"A batch of virus is expensive, but it doesn't compare to the cost of an additional clinical trial or comparability study triggered by a change in the manufacturing process late in development," says Lange. He suggests that partnering with a viral vector contract development and manufacturing organization (CDMO) helps you navigate the challenges with a focus on the science while leveraging their regulatory expertise to execute "right the first time" and avoid expensive mistakes along the way into the clinic and toward commercialization.

Scientists can overcome technical issues like low yields, impurities, cytotoxicity, and viral vector immunogenicity with many technologies developed for quality control and standardization. Marwan Alssaraj, Biopharma Segment Manager from Bio-Rad, reports that a robust quality control strategy includes regularly testing raw materials, intermediates, and final products.

"Multiple technologies are available to detect residual content and determine vector copy number during the production phase," Alssaraj adds, pointing out that Droplet Digital PCR (ddPCR) technology offers ultra-sensitive and absolute quantification of virus copy numbers by partitioning the sample into thousands of individual reactions, enabling precise quantification of the target DNA.

One-size-fits-all viral vector production?

The revolutionary aspect of the new viral vector platform production lies in its one-size-fits-all concept, where a single production system can manufacture various types of viral vectors.

Although this concept is attractive, it comes with challenges that must be overcome. Viral vector production depends on many factors, from vector type to quality control strategy, scale-up considerations, and regulatory requirements.

Experts are hesitant about this approach becoming a reality. According to Bio-Rad Senior Applications Manager Angelica Olcott, "Designing viral vectors for therapeutic gene delivery requires meticulous development, ensuring precise manufacturing under strict controls." Ensuring all these aspects for different vectors using the same production system may be challenging.

Lange additionally advises that "even within a single vector type, differences in process inputs can have outsize impacts on process outputs, impacting everything from cell viability in the upstream to final product quality."

Therefore, the optimal production system depends on your specific objective rather than a single, universally superior method that covers all criteria.

References

1. Philippidis, A. Making History with the 1990 Gene Therapy Trial. GEN - Genetic Engineering and Biotechnology News (2016).

2. Center for Biologics Evaluation and Research. KYMRIAH (tisagenlecleucel). FDA (2022).

3. Bulcha, J. T., Wang, Y., Ma, H., Tai, P. W. L. & Gao, G. Viral vector platforms within the gene therapy landscape. Sig Transduct Target Ther 6, 53 (2021).

4. Li, X. et al. Viral Vector-Based Gene Therapy. Int J Mol Sci 24, 7736 (2023).

5. Center for Biologics Evaluation and Research. Approved Cellular and Gene Therapy Products. FDA (2023).

6. Viral-vector therapies at scale: Today's challenges and future opportunities | McKinsey.