are tools commonly used by molecular biologist
s to deliver genetic material
s. This process can be performed inside a living organism ( in vivo
) or in cell culture
( in vitro
es have evolved specialized molecular mechanisms to efficiently transport their genome
s inside the cells they infect. Delivery of gene
s by a virus is termed transduction
and the infected cells are described as transduced. Molecular biologist
s first harnessed this machinery in the 1970s. Paul Berg
used a modified SV40
virus containing DNA from the bacteriophage λ
to infect monkey kidney
cells maintained in culture.
In addition to their use in molecular biology research, viral vectors are used for gene therapy
and the development of vaccine
Key properties of a viral vector
Viral vectors are tailored to their specific applications but 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. Such a virus can efficiently infect cells but, once the infection has taken place, requires a helper virus to provide the missing proteins for production of new virions.
- Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it infects.
- 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 and is avoided in their design.
- 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.
- Identification: Viral vectors are often given certain genes that help identify which cells took up the viral genes. These genes are called Markers. A common marker is antibiotic resistance to a certain antibiotic. The cells can then be isolated easily as those that have not taken up the viral vector genes do not have antibiotic resistance and so cannot grow in a culture with antibiotics present.
Viral vectors were originally developed as an alternative to transfection
of naked DNA
for molecular genetics
experiments. Compared to traditional methods such as calcium phosphate precipitation
can ensure that nearly 100% of cells are infected without severely affecting cell viability. Furthermore, some viruses integrate
into the cell genome
facilitating stable expression.
coding genes can be expressed
using viral vectors, commonly to study the function of the particular protein. Viral vectors, especially retroviruses, stably expressing marker gene
s such as GFP
are widely used to permanently label cells to track them and their progeny, for example in xenotransplantation
experiments, when cells infected in vitro
are implanted into a host animal.
Gene insertion is cheaper to carry out than gene knockout
. But as the silencing is sometimes non-specific and has off-target effects on other genes, it provides less reliable results. Animal host vectors also play an important role.
Gene therapy is a technique for correcting defective genes responsible for disease development. In the future, gene therapy
may provide a way to cure genetic disorder
s, such as severe combined immunodeficiency
, cystic fibrosis
or even haemophilia A
. Because these diseases result from mutation
s in the DNA sequence for specific genes, gene therapy trials have used viruses to deliver unmutated copies of these genes to the cells of the patient's body. There have been a huge number of laboratory successes with gene therapy. However, several problems of viral gene therapy must be overcome before it gains widespread use. Immune response
to viruses not only impedes the delivery of genes to target cells but can cause severe complications for the patient. In one of the early gene therapy trials in 1999 this led to the death of Jesse Gelsinger
, who was treated using an adenoviral vector.
Some viral vectors, for instance gamma-retroviruses, insert their genomes at a seemingly random location on one of the host chromosome
s, which can disturb the function of cellular genes and lead to cancer. In a severe combined immunodeficiency retroviral gene therapy
trial conducted in 2002, four of the patients developed leukemia as a consequence of the treatment; three of the patients recovered after chemotherapy. Adeno-associated virus-based vectors
are much safer in this respect as they always integrate at the same site in the human genome.
Viruses expressing pathogen
proteins are currently being developed as vaccine
s against these pathogens, based on the same rationale as DNA vaccines
s recognize cells infected with intracellular parasite
s based on the foreign proteins produced within the cell. T cell immunity
is crucial for protection against viral infections and such diseases as malaria
. A viral vaccine induces expression of pathogen proteins within host cells similarly to the Sabin Polio vaccine
and other attenuated vaccine
s. However, since viral vaccines contain only a small fraction of pathogen genes, they are much safer and sporadic infection by the pathogen is impossible. Adenoviruses
are being actively developed as vaccines.
Types of viral vectors
es are one of the mainstays of current gene therapy approaches. The recombinant retroviruses such as the Moloney murine leukemia virus
have the ability to integrate into the host genome in a stable fashion. They contain a reverse transcriptase
that allows integration into the host genome
. They have been used in a number of FDA-approved clinical trials such as the SCID-X1
Retroviral vectors can either be replication-competent or replication-defective. Replication-defective vectors are the most common choice in studies because the viruses have had the coding regions for the genes necessary for additional rounds of virion replication and packaging replaced with other genes, or deleted. These virus are capable of infecting their target cells and delivering their viral payload, but then fail to continue the typical lytic pathway that leads to cell lysis and death.
Conversely, replication-competent viral vectors contain all necessary genes for virion synthesis, and continue to propagate themselves once infection occurs. Because the viral genome for these vectors is much lengthier, the length of the actual inserted gene of interest is limited compared to the possible length of the insert for replication-defective vectors. Depending on the viral vector, the typical maximum length of an allowable DNA insert in a replication-defective viral vector is usually about 8–10 kB. While this limits the introduction of many genomic sequences, most cDNA
sequences can still be accommodated.
The primary drawback to use of retroviruses such as the Moloney retrovirus involves the requirement for cells to be actively dividing for transduction
. As a result, cells such as neurons
are very resistant to infection and transduction by retroviruses.
There is concern that insertional mutagenesis
due to integration into the host genome
might lead to cancer
. This concern remained theoretical until gene therapy for ten SCID-X1
patients using Maloney murine leukemia virus
resulted in two cases of leukemia caused by activation of the LMO2 oncogene
due to nearby integration of the vector.
es are a subclass of Retroviruses. They are sometimes used as vectors for gene therapy
thanks to their ability to integrate into the genome
of non-dividing cells, which is the unique feature of Lentiviruses as other Retroviruses can infect only dividing cells. The viral genome in the form of RNA
when the virus enters the cell to produce DNA
, which is then inserted into the genome at a random position (recent findings actually suggest that the insertion of viral DNA is not random but directed to specific active genes and related to genome organisation) by the viral integrase enzyme
. The vector, now called a provirus
, remains in the genome and is passed on to the progeny of the cell when it divides. The site of integration is unpredictable, which can pose a problem. The provirus
can disturb the function of cellular genes and lead to activation of oncogene
s promoting the development
, which raises concerns for possible applications of lentiviruses in gene therapy. However, studies have shown that lentivirus vectors have a lower tendency to integrate in places that potentially cause cancer than gamma-retroviral vectors. More specifically, one study found that lentiviral vectors did not cause either an increase in tumor incidence or an earlier onset of tumors in a mouse strain with a much higher incidence of tumors. Moreover, clinical trials that utilized lentiviral vectors to deliver gene therapy for the treatment of HIV experienced no increase in mutagenic or oncologic events.
For safety reasons lentiviral vectors never carry the genes required for their replication. To produce a lentivirus, several plasmid
s are transfected
into a so-called packaging cell line
, commonly HEK 293
. One or more plasmids, generally referred to as packaging plasmids, encode the virion protein
s, such as the capsid
and the reverse transcriptase
. Another plasmid contains the genetic material to be delivered by the vector. It is transcribed
to produce the single-stranded RNA viral genome and is marked by the presence of the ψ
(psi) sequence. This sequence is used to package the genome into the virion.
As opposed to lentiviruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division. This limits their use in basic research, although adenoviral vectors are still used in in vitro
and also in vivo
experiments. Their primary applications are in gene therapy
. Since humans commonly come in contact with adenoviruses
, which cause respiratory, gastrointestinal and eye infections, majority of patients have already developed neutralizing antibodies
which can inactivate the virus before it can reach the target cell. To overcome this problem scientists are currently investigating adenoviruses
that infect different species to which humans do not have immunity.
Adeno-associated virus (AAV) is a small virus that infects humans and some other primate species. AAV is not currently known to cause disease, and causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. Moreover, AAV mostly stays as episomal
(replicating without incorporation into the chromosome); performing long and stable expression. These features make AAV a very attractive candidate for creating viral vectors for gene therapy. However, AAV can only bring up to 5kb which is considerably small compared to AAV's original capacity.
Furthermore, because of its potential use as a gene therapy vector, researchers have created an altered AAV called self-complementary adeno-associated virus
(scAAV). Whereas AAV packages a single strand of DNA and requires the process of second-strand synthesis, scAAV packages both strands which anneal together to form double stranded DNA. By skipping second strand synthesis scAAV allows for rapid expression in the cell. Otherwise, scAAV carries many characteristics of its AAV counterpart.
Challenges in application
The choice of a viral vector
to deliver genetic
material to cells comes with some logistical problems. There are a limited number of viral vectors available for therapeutic use. Any of these few viral vectors can cause the body to develop an immune response
if the vector is seen as a foreign invader. Once used, the viral vector cannot be effectively used in the patient again because it will be recognized by the body. If the vaccine
or gene therapy
fails in clinical trials
, the virus can’t be used again in the patient for a different vaccine or gene therapy in the future. Pre-existing immunity
against the viral vector could also be present in the patient rendering the therapy ineffective for that patient. It is possible to counteract pre-existing immunity when using a viral vector for vaccination
with a non-viral DNA vaccine
, but this method presents another expense and obstacle in the vaccine distribution process. Pre-existing immunity may also be challenged by increasing vaccine dose or changing the vaccination
route. Some shortcomings of viral vectors (such as genotoxicity and low transgenic expression) can be overcome through the use of hybrid vectors