By Patti Cuevas and Peiqing Zhang

What is a viral vector?

Viruses are infectious agents that can only replicate inside of living cells. This trait is used by molecular biologists for delivery of genetic materials into cells. Viral vectors are also explored for use in cell and gene therapy and as a basis for prophylactic and therapeutic vaccines.

How do viral vectors work?

In gene therapy, viral vectors can be used for delivery of functional genes to replace defective genes to cure genetic disorders. As a vaccine platform, viral vectors can be used to express and present pathogenic antigens to induce an immune response by mimicking a natural infection. Viral vectors can also be used in oncolytic therapies to specifically target and kill tumor cells.

Viral vector systems

Although tailored to their specific applications, viral vectors share some key attributes. Vectors should be modified to provide safe handling (i.e., no production of new virions in host) and low toxicity (i.e., no effect of the physiology of the normal host cell). They should also be stable with no rearrangement of the genome. And for manufacturing, it is important that the viral vector is easily quantified and that it lends itself to large-scale production. Examples of viral vector systems are adeno-associated virus, lentivirus, gamma retrovirus, adenovirus, and herpes simplex virus.

Adeno-associated virus (AAV)

AAV is a single-stranded DNA virus that can infect both actively dividing and nondividing cells of humans and some other primates. It can integrate into the genome of the host cell but mostly, as is true with adenovirus, AAV replicates without incorporating its genome into the host cell chromosome. As opposed to adenovirus, which is a larger virus that can deliver DNA inserts of up to 36 kb, AAV is a small virus that can only deliver smaller inserts of up to 5 kb. AAV is the most common viral vector for in vivo gene therapy, with a handful of approved therapies on the market and more than 200 in clinical trials (1).

Learn why AAV is suitable for gene therapy applications.

Explore Cytiva resources for AAV development and manufacturing.

Lentivirus

Lentiviruses are a subclass of retroviruses, but in contrast to other retroviruses, lentiviruses can integrate into the genome of nondividing cells. As retrovirus vectors, lentivirus vectors never include genes for replication. Lentivirus production therefore requires propagation in cell lines such as HEK293 cells transfected with plasmids that encode the virion proteins. Lentiviruses are commonly used to transfer the chimeric antigen receptor (CAR) gene to primary T cells for CAR T cell therapy, due to their ability to achieve high rates of transduction and long-term stable transgene expression. They are also used to genetically engineer hematopoietic stem cells (HSCs) for ex vivo gene therapies.

Explore Cytiva resources for lentiviral vector development and manufacturing.

Gamma retrovirus

Recombinant retroviruses, including gamma retroviruses, have the ability to stably integrate into the host genome. These viruses express a reverse transcriptase to copy their RNA genome into DNA and an integrase to integrate the DNA copy into the genome of the host cell. Typically, replication-defective retroviruses are used in medicine, as these viruses can infect and deliver their viral genome, but they fail to lyse and kill the host cell. Retroviruses, however, can only integrate into the genome of actively dividing cells (with the exception of lentiviruses, as noted previously). Hence, many cells (e.g., neurons) are resistant to retrovirus infection and integration. Gamma retroviruses are used in CAR T cell therapies but aren’t as common as lentiviruses for this application.

Adenovirus

Adenoviruses are double-stranded DNA viruses that replicate in the cell nucleus of vertebrates. In contrast to retroviruses and lentiviruses, adenoviruses don’t integrate into the genome of the host cell. Adenoviruses allow foreign DNA to easily be introduced into their DNA, and they can be propagated in several cell types. In addition, adenoviruses have been shown to induce a broad immune response, including cytotoxic T cells. Hence, adenovirus is one of the most explored viral vectors for use in vaccines against infectious diseases and in oncolytic therapies. It has also been used in the development of therapeutic vaccines, and in gene therapies.

A number of clinical trials for cancer use human adenovirus as a delivery vector, and one gene therapy drug packaged with HAdV5 is approved for use in China. When these viral vectors are used clinically as oncolytic therapies, they can replicate. However, for other applications it’s important to ensure low levels of replication-competent adenovirus (RCA), which bears the risk of an unintended viral spread and host inflammation response (2).

Learn about our cell line that minimizes RCA.

Explore Cytiva resources for adenoviral vector development and production.

Herpes simplex virus

Herpes simplex virus (HSV) is an enveloped virus that can accommodate large transgene cassettes. HSV-1 has a strong tropism for neurons. One challenge is that it may elicit a strong immune response. A live, recombinant, replication-deficient HSV-1 viral vector is used in an oncolytic gene therapy applied topically to treat skin wounds.

Viral vectors in gene therapy

The five viruses described in this article are all used to some extent in gene therapy. Table 1 provides a summary of the viral characteristics.

 

Table 1. Viruses used for gene therapy

Parameter AAV Lentivirus Gamma retrovirus  Adenovirus Herpes simplex virus 
In vivo or ex vivo? In vivo Ex vivo  Ex vivo  In vivo  In vivo
Therapy type Viral gene therapy Gene-modified cell therapy*
 Gene-modified cell therapy  Viral gene therapy/ oncolytic virus  Oncolytic virus
 Coat Naked   Enveloped Enveloped   Naked  Enveloped
 Host genome interaction Integrating/non-integrating   Integrating Integrating   Non-integrating Integrating/non-integrating 
 Transgene expression Potentially long lasting   Long lasting  Long lasting Transient   Not applicable
 Approximate size (nm) 25   120  100  90  150

*Also being explored for viral gene therapy.

 

Viral vector vaccines

Viral vector vaccines are one option that avoids delivering the target pathogen itself. Instead, the viral vector delivers the genetic information to make the antigen of interest, usually a harmless part of the pathogen. Inside a vaccinated individual, the virus infects cells and produces large quantities of the antigen, which is designed to elicit an immune response and generate protective antibodies against future natural exposure. Adenovirus, vesicular stomatitis virus, and vaccinia virus are among the viruses developed into viral vectors. They are modified to remove disease-causing genes and, in some cases, to be defective in self-replication. Viral vectors have been used to develop and produce vaccines for infectious diseases such as COVID-19 and Ebola.

Viral vector manufacturing

Viral vectors have demonstrated their utility in numerous market-approved therapeutics and continue to feature heavily in hundreds of clinical trials. The growing number of potential indications and affected populations is driving the interest in developing manufacturing processes that are both scalable and cost-efficient. The global market value for viral vectors is projected to reach 5.5 billion USD by 2035 (3).

For regulatory compliance, products intended for therapeutic use should be well characterized and manufactured to high purity, efficacy, and safety, and high levels of good manufacturing practice (GMP) compliance should be met. The continuous development of recombinant viral vectors expands the commercial product pipeline, prompting the use of viral vector manufacturing platforms.

There are many points to consider during process development and vector production. Transient transfection of adherent mammalian cells is a common starting point, as it’s quick and accessible. Multiple plasmids are used to deliver all required components for viral replication plus the gene of interest (GOI). Processes initiated in 2D flatware can be scaled up by increasing the number of vessels; however, the footprint is large and manual labor is high as are operator-to-operator variability and contamination risk. A more suitable option for GMP environments and large-scale manufacturing is single-use bioreactors designed for adherent growth. These systems automate manual tasks, allow control over variability, and support closed processing. A bioreactor platform with appropriately sized units allows development at bench scale up to clinical and commercial production.

With substantial optimization and suitable equipment, transient transfection can be used at clinical scale. However, processes are often moved into cells adapted for suspension, as these can be grown to high density in single-use stirred-tank bioreactors. Packaging cell lines, with all required components except the GOI, and producer cell lines, with all components stably integrated, provide simplified manufacturing and support robust vector production batch after batch. They also support lower cost of goods (COGs) manufactured, as they reduce the number of expensive plasmids and reduce or eliminate the transfection reagent. Some so-called producer cell lines still require addition of adeno helper virus, which adds cost and is a contaminant that must be removed later.

Equally as important as the cell line and upstream process are the filtration and chromatography steps that will remove cellular debris and other contaminants such as host cell protein (HCP) and host cell DNA (hcDNA). A particular challenge with AAV is obtaining a large percentage of full capsids, which contain all genetic elements introduced to the cells. Empty and partial capsids, which include no or some DNA, respectively, must be removed to specified levels while still maintaining a good yield of full capsids. Anion exchange chromatography with optimized format and protocol is currently the main technique to perform this challenging separation effectively. A combination of single-use and reusable technologies can be used to achieve the overall manufacturing goals.

One aspect that is often overlooked when developing a process is the importance of analytics. Without appropriate and complementary methods that are reproducible, it isn’t possible to determine how the developed process is working or what the quantity and quality of the purified product are. Technologies such as surface plasmon resonance offer an attractive option for reproducible AAV titer analysis.

There are benefits to having a manufacturing platform instead of individual pieces of equipment. The necessary process equipment is integrated end to end ― from cell culture expansion systems to seed and production bioreactors and purification equipment through formulation and aseptic filling. A configurable, scalable process train allows process developers and manufacturing staff to start small with their production requirements and grow as needed. Automation enhances the process line with centralized data management and streamlined production in an environment ready for 21 CFR Part 11 compliance.

Learn about our process train for biopharma manufacturing.

  1. US National Institutes of Health, National Library of Medicine, National Center for Biotechnology Information. ClinicalTrials.gov. Accessed February 28, 2024.
  2. Xie L, Han Y, Liu Y et al. Viral vector-based cancer treatment and current clinical applications. MedComm-Oncology. 2023;29 October. https://doi.org/10.1002/mog2.55.
  3. Roots Analysis. Viral Vector Manufacturing Market (6th Edition), Industry Trends and Forecasts Till 2035. https://www.rootsanalysis.com/reports/viral-vector-manufacturing-market/toc.html.