Q&A: Overcoming manufacturing challenges with lentiviral vectors
EPM chats with Pratima Cherukuri, chief scientific officer, Genezen, who shares the considerations in the development and manufacture of lentiviral vectors, and highlights some of the challenges organisations may face as they look to bring these potentially life saving therapies to market.
Key insights:
- Lentiviruses' advancement in C>s can be seen in the success of CAR-T therapies, representing 48% of all technology in the genetically modified cell therapy development pipeline.
- One of the most prevalent challenges that developers and manufacturers are facing in lentivirus production currently is the desire for stable producer cell lines.
- LV developers can adapt adherent cell lines to suspension systems, which are associated with ease of scaling and high levels of control over culture parameters.
Q. As a tool for introducing genetic material to patient cells in cell and gene therapy (C>) applications, what key considerations need to be made when selecting a viral vector?
A. As different virus types have unique characteristics and properties, not all viral vectors will be suitable for all applications, even with our ability to manipulate and modify them genetically. Consequently, different viral vectors will occupy different “niches” in the C> space.
This can be seen when comparing adeno-associated viral vectors (AAVs), lentiviral vectors (LVs), and gammaretroviral vectors (RVs). AAVs are small non-integrating viruses that are mildly immunogenic, making them ideal tools for in vivo treatments. However, because of their smaller packaging capacity, AAVs cannot be used for applications requiring large genetic material needs like CRISPR genome editing.
On the other hand, LVs and RVs can carry a much larger cassette and can stably integrate their genetic material into the target cell genome - a property not shared by AAVs. This integration enables long-term gene expression, preventing the need for frequent re-treatment. In comparison to AAVs, RV and LV applications are also best-suited to ex vivo applications, like chimeric antigen receptor (CAR) T and natural killer (NK) cell therapies.
The choice of viral vector will therefore be project-specific, and developers will need to carefully consider the characteristics and properties of each to make the best call for the C> application.
Q. What role have lentiviral vectors played in the advancement of cell C>s to date?
A. The role of lentiviruses in the advancement of C>s can be seen in the success of CAR-T therapies. These revolutionary treatments typically use LVs or RVs to introduce CAR genes to patients’ T cells, allowing the specific recognition and destruction of cancer cells. Since the first CAR-T therapy was approved by the FDA in 2017, five more have been introduced to the market. Their success has meant that CAR-T cell therapies remain the most common technology in the genetically modified cell therapy development pipeline, representing 48%.
In part, the growing preference for the use of LVs in C>s over the past decade has been driven by safety concerns around the alternative gene delivery tool, gammaretroviruses. RVs have the propensity to integrate genes near oncogene promoters in target cells, which can cause gene misregulation and lead to normal cells becoming cancerous. This caused a trend in the C> space for developers to move away from RVs to LVs.
Recently, many CAR-T therapy developers have shown a preference for the use of RVs due to the risk of oncogenesis being less of a concern when using cells that won’t proliferate, like mature T cells. Unfortunately, these changing trends have meant it is now challenging to find those with suitable expertise in RV production to provide support in development and manufacturing.
Q. What do you consider to be the most difficult challenges in lentiviral vector development and manufacturing currently?
A. One of the most prevalent challenges in lentivirus production that developers and manufacturers are facing currently is the desire for stable producer cell lines.
Typically, methodologies for LV production will be based on the overexpression of transiently transfected plasmid DNA in highly transfectable cell lines like HEK293. As transient transfection by nature only facilitates short-term expression of viral vector genes, transfection methods must be carried out every time a new batch is needed. This can be costly, especially considering the high price of clinical-grade transfection reagents and plasmids.
By generating stable cell lines (where the genes for LV production are integrated into the producer cell genome), long-term expression can be achieved, offering decreased batch-to-batch variability, greater quality particles, and reduced reagent and plasmid costs.
The generation of stable cell lines for LV production is not easy because the integration of potentially cytotoxic transgenes into the producer cell genome can cause premature cell death, impacting yield. Therefore, expertise is needed to carefully modify these cell lines to allow precisely controlled expression of cytotoxic proteins.
Q. In your opinion, which technologies have most significantly improved the lentiviral vector manufacturing processes?
A. Fixed-bed bioreactors have had a significant impact on lentiviral vector production, especially considering the ability to scale projects to clinical and commercial levels.
Most producer cell lines used for LV production are adherent, requiring an adhesive surface for growth and offering high titers. In the past, LV manufacturers had to rely on traditional 2D adherent systems like stackable cell culture chambers. As projects scaled, increasing numbers of chambers would be needed, occupying large amounts of facility space, and requiring copious manual handling activities and the associated higher risk of contamination.
Depending on the cell line, LV developers can adapt adherent cell lines to suspension systems, which are associated with ease of scaling and high levels of control over culture parameters. This is typically arduous and suspension cultures generally can’t achieve titers as high as their adherent counterparts.
The introduction of fixed-bed bioreactors has offered an attractive alternative to the need to rely on 2D adherent systems or adaption to suspension systems. With a 3D matrix that provides a high adherent surface-to-volume ratio, automated fixed-bed bioreactors can be used to scale upstream processing activities while requiring less physical space and manual handling activities as compared with traditional 2D systems.
Q. What advice would you give to developers and manufacturers that are beginning their lentiviral vector project journey?
A. We have seen how quickly advances in our understanding of molecular biology and technologies can help to evolve therapeutic areas from the success of global inoculation efforts with a relatively new tool: mRNA vaccines. With this in mind, my advice would be to find support from those at the forefront of advances in the C> space that stay abreast of innovations and regulatory changes.
Our understanding of C>s has expanded dramatically over the past decade and as a result, regulatory bodies have had to respond rapidly to introduce guidance for developers and manufacturers taking on this new challenge. As our knowledge grows and we see further technological advances, we can anticipate this guidance to be adapted and altered. With support from a contract development and manufacturing organisation (CDMO) that has transparent and open communications with regulators, viral vector developers can ensure their project will remain compliant with future regulatory changes.
By partnering with lentivirus development and manufacturing experts with close connections to innovative academic departments or those stemming from these environments, you can be assured that they will be well aware of advancements in the area.