For cell types not amenable to lipid-mediated transfection, viral vectors are often employed. Virus-mediated transfection, also known as transduction, offers a means to reach hard-to-transfect cell types for protein overexpression or knockdown, and it is the most commonly used method in clinical research (Glover et al., 2005; Pfeifer and Verma, 2001). One of the main advantages of viral delivery is that the process can be performed inside a living organism (in vivo) or in cell culture (in vitro) with gene delivery efficiencies approaching 95–100%.
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Key properties of viral vectors
Viral vectors are tailored to their specific applications, but must generally share a few key properties:
- Safety: Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. This usually involves the deletion of a part of the viral genome critical for viral replication, allowing the virus to efficiently infect cells and deliver the viral payload, but preventing the production of new virions in the absence of a helper virus that provides the missing critical proteins. However, an ongoing safety concern with the use of viral vectors is insertional mutagenesis, in which the ectopic chromosomal integration of viral DNA either disrupts the expression of a tumor-suppressor gene or activates an oncogene, leading to the malignant transformation of cells (Glover et al., 2005).
- Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it infects. This is especially important in studies requiring gene delivery in vivo, because the organism will develop an immune response if the vector is seen as a foreign invader (Nayak and Herzog, 2009).
- Stability: Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector. Therefore, unstable vectors are usually avoided.
- Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell types as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell. Viruses modified in this manner are said to be pseudotyped.
- Selection: Viral vectors should contain selectable markers, such as resistance to a certain antibiotic, so that the cells that have taken up the viral vector can be isolated
Common viral vectors
Adenoviruses
Adenoviruses are DNA viruses with broad cell tropism that can transiently transduce nearly any mammalian cell type. The adenovirus enters target cells by binding to the Coxsackie/Adenovirus receptor (CAR) (Bergelson et al.,1997). After binding to the CAR, the adenovirus is internalized via integrin-mediated endocytosis followed by active transport to the nucleus, where its DNA is expressed episomally (Hirata and Russell, 2000). Although adenoviral vectors work well for transient delivery in many cell types, for some difficult cell lines such as non-dividing cells and for stable expression, lentiviral vectors are preferred. The packaging capacity of adenoviruses is 7–8 kb.
Retroviruses
Retroviruses are positive-strand RNA viruses that stably integrate their genomes into host cell chromosomes. When pseudotyped with an envelope that has broad tropism, such as vesicular stomatitis virus glycoprotein (VSV-G), these viruses can enter virtually any mammalian cell type. However, most retroviruses depend upon the breakdown of nuclear membrane during cell division to infect cells and are thus limited by the requirement of replicating cells for transduction. Other disadvantages of retroviruses include the possibility of insertional mutagenesis and the potential for the activation of latent disease. Like adenoviruses, retroviruses can carry foreign genes of around 8 kb.
Lentiviruses
Lentiviruses are a subgroup of the retrovirus family; as such, they can integrate into the host cell genome to allow stable, long-term expression (Anson, 2004). In contrast to other retroviruses, lentiviruses are more versatile tools as they use an active nuclear import pathway to transduce non-dividing, terminally differentiated cell populations such as neuronal and hematopoietic cells.
Adeno-associated viruses
Adeno-associated viruses are capable of transducing a broad range of dividing and non-dividing cells types, but they require coinfection with a helper virus like adenovirus or herpes virus to produce recombinant virions in packaging cells. This causes difficulties in obtaining high quality viral stocks that are free of helper viruses. Furthermore, adeno-associated viruses have only limited packaging capacity of up to 4.9 kb. On the other hand, adeno-associated viruses show low immunogenicity in most cell types, and they have the ability to integrate into a specific region of the human chromosome, thereby avoiding insertional mutagenesis.
Other viral vector systems that can be used for overexpression of proteins include vectors based on baculovirus, vaccinia virus, and herpes simplex virus. While baculoviruses normally infect insect cells, recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by including a dominant selectable marker in the baculoviral vector, cell lines can be derived that stably express recombinant genes (Condreay et al., 1999). Vectors based on vaccinia virus can be used for introducing large DNA fragments into a wide range of mammalian cells. However, cells infected with vaccinia virus die within one or two days, limiting this system to transient protein production. Herpes simplex viruses are a class of double-stranded DNA viruses that infect neurons.
| Viral System | Size | DNA insert size | Max titer (particles/mL) | Infection | Expression | Drawbacks |
|---|---|---|---|---|---|---|
| Adenovirus | 36 kb (dsDNA) | 8 kb | 1 × 1013 | Dividing and non-dividing cells | Transient | Elicits strong antiviral immune response |
| Retrovirus | 7–11 kb (ssRNA) | 8 kb | 1 × 109 | Dividing cells | Stable | Insertional mutagenesis potential |
| Lentivirus | 8 kb (ssRNA) | 9 kb | 1 × 109 | Dividing and non-dividing cells | Stable | Insertional mutagenesis potential |
| Adeno-associated virus | 8.5 kb (ssDNA) | 5 kb | 1 × 1011 | Dividing and non-dividing cells | Stable; site-specific integration | Requires helper virus for replication; difficult to produce pure viral stocks |
| Baculovirus | 80–180 kb (dsDNA) | no known upper limit | 2 × 108 | Dividing and non-dividing cells | Transient or stable | Limited mammalian host range |
| Vaccinia virus | 190 kb (dsDNA) | 25 kb | 3 × 109 | Dividing cells | Transient | Potential cytopathic effects |
| Herpes simplex virus | 150 kb (dsDNA) | 30–40 kb | 1 × 109 | Dividing and non-dividing cells | Transient | No gene expression during latent infection |
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