Since the 1990s, cell and gene therapy research has expanded to include an increasing number of FDA-approved therapies for conditions ranging from cancer to rare hereditary diseases. The versatility of cell and gene therapy lies in the ability to modify and introduce any gene sequence into cells. Manufacturing enough cell and gene therapy products and transferring those products safely across cell membranes relies on critical early steps in therapeutic development.
Download this Explainer Article from Mirus to learn more about how scientists produce safe and effective products for cell and gene therapy.
Explained: HOW DO SCIENTISTS PRODUCE SAFE AND EFFECTIVE PRODUCTS FOR CELL AND GENE THERAPY?
The early steps of viral vector production have a profound effect on cell and gene therapy success.
In the early 1990s, cell and gene therapy transitioned from concept to reality. Today, there are an increasing number of FDA-approved cell and gene therapy products in the United States, and thousands more in clinical trials for many conditions that range from cancer to rare hereditary diseases. The versatility of cell and gene therapy lies in the ability to modify and introduce any gene sequence. Transporting therapeutic sequences into cells can be achieved with viral vectors such as lentiviruses and adeno-associated viruses (AAV), which natively excel at delivery of genetic information. Lentiviral vectors are capable of enacting permanent changes to cellular genomes, and are typically used to modify cells outside of the body and are then reintroduced into patients. In contrast, AAVs are the vector of choice for direct administration to patients because they do not typically integrate into cellular genomes. AAVs are also less immunogenic than other viruses, making them ideal viral carriers for gene therapy.
How do scientists make viruses for cell and gene therapy?
To safely work with viruses, researchers typically separate the viral genome into multiple plasmids. One plasmid contains the therapeutic gene sequence--the genetic ‘payload’--which is packaged into the viral vector. The other plasmids encode viral genes required for packaging and the structural proteins that comprise the virus. By introducing select mutations and separating viral machinery, the recombinant virus that is produced will be replication incompetent (1).
Reassembling recombinant viral vectors is not like reassembling a puzzle. The separated viral components need to be assembled within specialized cell lines that are uniquely susceptible to plasmid transfection and sturdy enough to withstand viral growth. Although scientists have used one such cell line, the human embryonic kidney 293 (HEK293) cell line, for plasmid transfection for decades, it still needs help taking up plasmids. Cell membranes naturally repel negatively charged plasmid DNA. This mechanism protects cells from taking up potential pathogens. To convince cells to take up plasmids, researchers use a chemical concoction called a transfection reagent that condenses and coats plasmid DNA in a positive charge, allowing it to more readily cross cell membranes.
Transfection reagents are commonly classified as being lipid or polymer-based. Lipid-based transfection reagents that form liposomes, while effective, can exert cytotoxic effects. Polymerbased transfection reagents typically do not form liposomes and may interfere less with cell membrane integrity (2). Some transfection reagents, such as the TransIT-VirusGEN® Transfection Reagent from Mirus Bio combine both lipids and polymers. Components in this transfection reagent complex with negatively charged plasmid DNA to form positively charged complexes, called lipopolyplexes. The lipopolyplexes associate favorably with cell membranes via electrostatic interactions to facilitate intracellular uptake of plasmids via endocytosis.
What steps can scientists take to produce high viral titers for gene and cell therapy?
Complex formation is a critical step in the transfection process. The time it takes for a transfection reagent to interact with plasmid DNA and form complexes, known as complexation time, correlates with complex size. Longer complexation times often yield larger transfection complexes, which can directly affect transfection efficiency. Transfection complexes range in size from 100 nm to sub-micrometer scale; the size requirement of the transfection complex will depend on the type of transfection reagent and specific application involved (3). Although larger complexes are generally thought to more easily interact with cell surfaces, leading to better cellular uptake, this is not true for all cell types (4). Choosing the best transfection reagent for each experiment relies on selecting the best match between the physical/chemical properties of the transfection complex and the endomembrane system of the target cell (3).
The TransIT-VirusGEN® Transfection Reagent has a broad complexation time, with effective transfection complexes for viral vector production in HEK293 cells being formed as soon as 15 minutes up to over an hour. This large window of time to form complexes is especially beneficial for large-scale transfections which may require more time to move huge volumes of materials from one vessel to another. The ratio of transfection reagent to DNA also affects complex size and growth kinetics. In general, more concentrated mixtures yield more quickly growing complexes. Therefore, most transfection protocols include recommended parameters for the ratio of transfection reagent to nucleic acid concentration that complement recommended complex formation time.
When scaling up the viral production process, researchers also need to consider the sheer amount of space to house large volumes of packaging and patient cells. A single dose of AAV-based gene therapy can require as many as 1016 viral genomes, necessitating transfection of several hundreds of liters of cells. Bioreactor capacity can range from the L to kL scale, and care must be taken in developing a strategy for seeding and expanding cells to the appropriate density for transfection or cell therapy for each size and type of bioreactor. The time and labor involved with every step is also amplified with increasing the size and number of transfections. Interestingly, the molecular dynamics of transfection may also differ for suspended cells in bioreactors because transfection complexes are less likely to attach to cell surfaces (5). However, many new transfection reagents, including the TransIT-VirusGEN® Transfection Reagent, efficiently transfect cells in suspension cultures.
How can reagents affect the safety of gene and cell therapy?
Growing cells in vitro can require unique culture conditions. Researchers often rely on serum as a source of transcription factors, hormones, lipids, and minerals to support continual growth and health of cultured cells. However, serum is usually an animalderived product which has a risk of carrying and transmitting pathogens.
Residual animal factors in serum, transfection reagents, or other ancillary products (i.e., materials used in the manufacturing process but not intended to be in the drug) are undesirable in cell and gene therapeutics. Animal byproducts, however, are not the only concern. The microbial world can also creep into cell culture reagents. An estimated one-third of all cell culture is contaminated with Mycoplasma, a genus of pathogenic bacteria that can affect cell physiology (6). Endotoxins released from bacterial cells may produce cytotoxic effects. If animal byproducts, Mycoplasma, or endotoxins infiltrate cell and gene therapy products they can produce deadly immunogenic responses in patients.
To mitigate this risk, researchers should use high quality reagents at every step in the cell and gene therapy production process, including transfection. The TransITVirusGEN® GMP Transfection Reagent and Kits undergo rigorous quality control and tests to ensure they are animal-free, Mycoplasma-free, and endotoxin-free so they are ready to be used in GMP-compliant drug manufacturing.
Establishing a clear path
The wide range of TransIT-VirusGEN® transfection products allows researchers to seamlessly transition from the same reliable transfection technology they used for small scale research and discovery to large scale viral production. This saves researchers the trouble of switching reagents mid-process and overhauling their transfection workflow. They can also rest assured that each transfection reagent has been rigorously tested to ensure optimal high-titer transfection on various viral vector production platforms as well as purity and quality. These uniquely developed transfection reagents have helped scientists characterize an RNA-based vaccine against SARS-CoV-2, uncover the role of unique chromatin dynamics for melanoma, and generate enough viral vector for a new large scale hemophilia gene therapy (7, 8, 9). Optimizing viral vector production streamlines the path to ground breaking discovery.
References
1. Markowitz, D., Goff, S., & Bank, A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. Virol J 62(4), 1120-1124 (1988).
2. Chong, Z. X., Yeap, K. S., & Ho, W. Y. Transfection types, methods and strategies: a technical review. Peer J 9, (2021).
3. Deliverance: understanding transfection complexes. Mirus Bio. https://www.mirusbio.com/blog/deliverance (2022).
4. Pezzoli, D., et al. Size matters for in vitro gene delivery: investigating the relationships among complexation protocol, transfection medium, size and sedimentation. Sci Rep 7, (2017).
5. Basiouni, S., Fuhrmann, H., & Schumann, J. High-efficiency transfection of suspension cell lines. Biotechniques 53, (2018).
6. Nikfarjam, L., & Farzaneh, P. Prevention and detection of mycoplasma contamination in cell culture. Cell J 13, 203-212 (2011).
7. Erasmus, J.H., et. al. An Alphavirus-derived replicon RNA vaccine induces SARS-CoV-2 neutralizing antibody and T cell responses in mice and nonhuman primates. Sci Transl Med 12, (2020).
8. Carcamo, S., et al. Altered BAF occupancy and transcription factor dynamics in PBAF-deficient melanoma. Cell Rep 39, 110637 (2022).
9. Global hemophilia B gene therapy trial. St. Jude Children’s Research Hospital. https://www.stjude.org/research/departments/hematology/b-global.html