Viral vectors are the principal gene delivery mechanism for gene therapy, but the technique is also used to improve cells used in research and biomanufacturing—the topic of this roundtable discussion.
Biocompare questioned five experts on the hows and whys of viral vectors: why use them, how to use them, and what to expect commercially. Our panel: Elier Paz, CEO of Canvax Biotech; Ken Rosser, vice president of business operations at CELL BIOLABS; Henry George, head of viral vector producer cell lines at MilliporeSigma; Cédric Sapet, Ph.D., head of life sciences R&D at OZ Biosciences; and Xavier J. de Mollerat du Jeu, director of R&D, cell biology for Thermo Fisher Scientific.
Q1: Why use viral vectors when simpler gene-transfer protocols exist?
Elier Paz: Viral transduction is the method of choice when stable transgene expression and short development time are needed. Using a lentiviral vector takes three to four weeks, compared with eight to twelve weeks using plasmids under antibiotic control.
Ken Rosser: Viral vectors are usually not the first choice, but some cells are difficult to permeate with traditional chemical and physical transfection methods. Using a viral vector can overcome these permeability issues by taking advantage of the virus' infectious nature.
Henry George: While vast improvements have occurred with transfection agents, some cell types are still difficult to transform due to low efficiency of DNA uptake. CHO-derived lines transfect at very low efficiency using chemical agents, while electroporation is difficult to scale. Selection of stably-integrated chromosomal events is another hurdle that is not easily met with chemical or physical transfection. Viral vectors work on the premise that most cell types can be infected with an appropriate viral particle, with stable gene integration approaching 100%.
Cédric Sapet: For bioproduction cells like HEK293 and CHO, viral vectors allow either transient or stable transduction. Another application where viral vector might be selected is for the transduction of primary cells expressing specific proteins like T-CAR.
Xavier J. de Mollerat du Jeu: Viruses are used for better efficiency and sustained expression of the transcript. For example, lentiviruses that have integrated their transgene into the host genome provide sustained expression of the gene of interest and provide an easy way to create stable cell lines. Although adeno-associated virus vectors don’t integrate in the genome, they also provide long duration of expression in non-dividing cells.
Q2: What are the most common viral agents and target cells?
Elier Paz: The most common viral agents are retrovirus and adenovirus for mammalian cells, baculovirus for insect cells, and phages for bacteria. Among retrovirus there are two main types: gamma retrovirus (e.g. MMLV-derived viruses) and lentivirus (e.g. HIV or FIV-derived viruses). For human drug development, the most commonly used cell lines are CHO, NS0, and HEK293 or derivatives of the above cell lines, while E. coli is the most common bacterial expression systems. The use of viral agents is still uncommon in bioproduction, but recent successes in gene therapy may speed-up the use of viral vectors for development of cell lines for bioproduction.
Ken Rosser: For basic research studies, the most common viral agents are adenovirus, adeno-associated virus (AAV), lentivirus, and retrovirus. Virtually any cell could be a target cell, depending on the reason for the experiment, including immortalized cancer cells and primary cells from virtually any tissue or organ.
Henry George: The viral particle envelope and/or capsid proteins typically dictate the type of receptor and hence cell type that the virus will bind to and therefore infect. While many different cell types can be transduced or infected with a carefully engineered envelope or capsid pseudotyped virion particle, for the large-scale production of recombinant proteins, typically those cell types are the more common and easy to grow HEK293, 293T, and CHOK1 cell lines.
Q3: What are the goals of viral vector transduction?
Elier Paz: I see a future for viral vectors in the generation of stable cell lines for producing toxic proteins.
Ken Rosser: Viral vectors introduce a specific gene into a target cell to see the effect of the expression of that gene. Engineering improvements into a cell is possible as well using a virus that integrates into the genome of the target cell, such as lentivirus or retrovirus.
Cédric Sapet: There are so many goals of viral vector transduction, for example improvement in cell characteristic leading to higher protein production or lower production cost. The starting point is that gene encoded by viral vector can integrate into the cell genome, which rapidly establishes a stably-transduced cell line expressing the gene of interest. The benefit of stable transduction is the gain in time, and that the line remains stably modified for a new production run.
Q4: How does the value chain for viral vectors proceed, from concept to end-use?
Elier Paz: Canvax Biotech produces viral vectors starting either from a gene sequence, or from a plasmid vector containing the full gene sequence. We also offer plasmid packaging kits for production of viral particles. One important question is the kind of cells that are going to be transduced at the end, for example CHO cells are not readily transduced with ecotropic retroviruses. The right virus should be determined between the CRO and the research group, as there is no universal solution.
Ken Rosser: CELL BIOLABS does not provide cloning or viral construction services. We sell plasmids that allow a researcher to clone in the gene of interest and package the viral particle containing that gene. The target cells depend more on their species and the type of virus being used (e.g. AAV, lentivirus, etc.) so generally our kits are fairly universal in targeting a variety of cells, with a few exceptions.
Henry George: In early days, academic labs produced viral vectors that were handed down from lab to lab with little to no quality control, and without well-developed protocols. Today commercial companies supply the tools and accessories for producing such viruses. Many companies also offer customized viral construction and production services, while others sell ready-to-use retroviruses carrying a variety of gene expression or gene editing platforms. One such example is MilliporeSigma Mission® shRNA library, which allows customers to order a viral vector carrying a specific shRNA construct against nearly any gene within the human and mouse genome. Platform systems consisting of bundled cell lines, viral packaging vectors, cell growth medium, protocols, etc., are also readily available from several commercial vendors.
Cédric Sapet: OZ Biosciences provides products for viral applications, from production of recombinant virus by transfection of plasmids (CaPO kit or Helix-IN transfection reagent), capture, concentration, and conservation of viruses (Mag4C) to viral enhancers (Lentiblast-chemical-based and ViroMag R/L-magnetic nanoparticles based). We also own a proprietary technology, Magnetofection, a tool for improving viral efficiency.
Xavier J. de Mollerat du Jeu: There are many ways. Users can purchase kits—we just recently launched the LV-MAX Lentiviral Production System, which allows users to produce their own lentivirus. We provide cells, media transfection enhancers, and detailed protocol to maximize production of lentiviruses. For vectors, most are commercially available or you can design them using a gene synthesis company like GeneART. Finally, we also offer services, through which we can provide ready-to-use viruses for diverse applications. Universities have viral core labs that can also provide viruses.
Q5: What should end-users look for in a viral vector kit, product, or service?
Elier Paz: When selecting a viral vector, users should ask what is the main problem to be solved and how viral vectors might help. If there is rationale for using viral vectors, then they should be tried because of their reliability.
Ken Rosser: There are many considerations when looking for a viral vector, because there is not one type of virus that is the best for all experiments. We generated a table to allow users to make an informed decision on the best viral vector for their situation [see table below].
Henry George: Several limitations arise with viral vectors. First, smaller virus systems like AAV, can carry a gene package limited to about 5K bases, whereas the retro and lentiviral systems can package up to 10 Kb of genetic material. While most lentiviral systems use a pseudotype envelope protein to ensure good transduction efficiency, systems like AAV allow more targeted infection by using capsids that recognize specific receptor proteins on different cell types. A sought-after characteristic is the production of high titer virion particles that can be immediately used for downstream applications without the need for additional purification or concentration. While most current platform systems can produce moderate titer levels, some refinement of both individual technique and processes are usually required in order to generate virus of high quality and titer. Finally, safety of the use of viruses plays a large role when using any biological system that can infect, integrate, and disrupt essential genes.
Cédric Sapet: End-users should look for high titers, high ratio of infectious particles to non-infectious particles, transduction efficiency, strong gene expression, and stability. End-users should always consider using enhancers such as Lentiblast or ViroMag R/L to improve efficiency, while using low viral doses (titers), which reduces cost.
Xavier J. de Mollerat du Jeu: Users should look for a virus with high transfection efficiency and minimal multiplicity of infection. Key information are titers (PCR-based or functional-based), full versus empty particles, P24 ELISA quantification, and ratios of different capsid protein.
Table: Checklist for viral vector selection. Table courtesy of Cell Biolabs.
Top Image: HEK 293 cells infected with AAV coded with Green Fluorescent Protein. Image courtesy of Cell Biolabs.