My Favorite Building Block   |   Aug 02, 2023

So Many Viral Options. How Do You Choose?

When planning a gene delivery experiment, one of the first decisions to make is whether you’re going to use a viral or non-viral delivery method. If you choose to go viral (ba-dum tss), there are still many options to choose from. Lentivirus? Self-inactivating MMLV? Gutless adenovirus? With so many viral vectors to choose from, how do you know which is best?

Virus or no virus?

Whether you should use viral or non-viral delivery is largely dependent on your target cells. Classic non-viral transfection methods include electroporation, injection of plasmids directly into cells, or use of transfection reagents but these strategies are largely limited to in vitro systems, a smaller range of cell types, and smaller scales. For instance, differentiated cells are often difficult to transfect due to a number of factors including cytoskeletal changes, reduced proliferation, and the increased presence of nucleases. More modern methods for transfection include lipid nanoparticles (LNPs) for introducing plasmids or mRNA through lipofection. This technology was utilized for IVT mRNA COVID-19 vaccines and shows great promise for other therapies, but its use has limitations including inability to target specific tissues, instability, and short duration of the delivered genetic material.

For cells that are difficult to transfect (e.g. most in vivo systems) or require long term expression of transgenes, viral vectors are an excellent choice. However, there are over a dozen different recombinant viral systems to choose from. Which system suits you and your experiment depends on your application, the size of your insert, and your cells.

When it’s forever…

After deciding that a specific viral system is for you, one of the first questions to ask is whether you want the genetic material to be permanently integrated into the target cell’s DNA or transiently expressed. As seen in the graphic below, options for recombinant viral systems with genome integration include lentivirus and gamma retroviruses. Lentivirus is a popularly used system in both research and clinical applications due to its broad tropism and ability to infect both dividing and non-dividing cells. Lentivirus is a popular tool for introducing transgenes in research and clinical settings and is the basis for a recently FDA-approved treatment for beta-thalassemia, Zynteglo.

Other retroviruses in the family of γ-retroviruses including MMLV (Moloney Murine Leukemia Virus) and MSCV (Murine Stem Cell Virus) are popular variants of retrovirus used in research applications but have some limitations. Primarily, they are only able to integrate their genetic cargo in actively dividing cells. In addition, they tend to produce lower viral titers, and have smaller carrying capacities: inserts must be less than 5.5 kb for MMLV and 6.1 kb for MSCV, compared to 6.4 kb for lentivirus. Additionally, MMLV and MSCV transgenes are driven by viral promoters, although a modified MMLV (self-inactivating MMLV, or SIN MMLV) allows for use of alternative promoters. MMLV is useful when generating iPSCs, while MSCV is ideal for transducing HSCs.

When it’s not…

Many experiments and applications do not require stable integration into the genome. These non-integrating viruses typically maintain their genetic material, including the insert, in an extra-chromosomal or episomal form. If the transduced cells continue to divide, the recombinant virus’s genome will dilute out over time. However, if the target cells are fully differentiated or if shorter-term expression is needed, these viruses are an excellent option.

Two popular options from this group are adenovirus and adeno-associated virus (AAV). Both viruses can infect both dividing and non-dividing cells, but they differ in many respects. The major strengths and weaknesses for each are listed below. Notably, development of all of these systems continues, hoping to further enhance their strengths or offset their weaknesses. For instance, modifications of adenovirus have been developed to expand tropism (chimeric Ad5/F35 adenovirus) and carrying capacity (gutless Ad5). For AAV, the hunt is on for new serotypes to enhance tissue specificity and transgene expression.

Advantages Disadvantages
Adenovirus
  • Larger carrying capacity (7.5-33 kb)
  • High expression levels
  • High immunogenicity
  • Broad tropism, but difficulty transducing some cells**
AAV
  • Very low immunogenicity
  • Flexibility in targeting with different serotypes
  • Long-lasting gene expression
  • Small carrying capacity (4.2 kb)
  • Lower expression levels

** Endothelia, smooth muscle, differentiated airway epithelia, peripheral blood cells, neurons, HSCs

Outside of these popular choices, other systems are attractive for applications like vaccines and oncolytic virus. Vesicular stomatitis virus (VSV), vaccinia virus (VACV), and herpes simplex virus (HSV) generally have high carrying capacities (11, 30, and 150 kb, respectively). Additionally, all have high immunogenicity, lending to strong potential for vaccine development and immune system activation. VSV has traditionally been used to study viral entry mechanisms, but in recent years recombinant VSV-based vaccines have gained greater interest. While VACV gained fame as a platform for a smallpox vaccine in the 1800s, many strains have been developed with various attenuations, oncolytic activities, and potential for vaccines for a variety of diseases. Similarly, the strong neurotropism of HSV has been utilized in various research areas including the study of neural connectivity, but areas of interest have expanded to cancer treatment and vaccine delivery.

There are many options for viral vectors, and limitless options for how to use them. Many of these systems can be combined with inducible, conditional, or expression editing systems. You can determine the most efficient and comprehensive approach by considering your application, target system and cells, and insert size. The graphic below provides an overview of popular viral vectors, and we are always on hand to help you find the best solution to advance your research.

Sources

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Mulia GE, Picanço-Castro V, Stavrou EF, Athanassiadou A, Figueiredo ML. Advances in the Development and the Applications of Nonviral, Episomal Vectors for Gene Therapy. Hum Gene Ther. 2021 Oct;32(19-20):1076-1095. doi: 10.1089/hum.2020.310. Epub 2021 Sep 20. PMID: 34348480; PMCID: PMC8819515.

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