Viral vector
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'''Viral vectors''' are a tool commonly used by molecular [[biologist]]s to deliver [[genetic material]] into [[cell (biology)|cell]]s. This process can be performed inside a living organism (''[[in vivo]]'') or in [[cell culture]] (''[[in vitro]]''). [[Virus]]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 (genetics)|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]] [[lambda phage|lambda]] to infect monkey [[kidney]] cells maintained in culture.<ref>Goff SP and Berg P. (1976) Construction of hybrid viruses containing SV40 and lambda phage DNA segments and their propagation in cultured monkey cells. ''Cell''. '''9''':695-705. {{Entrez Pubmed|189942}}</ref>
==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 [[protein]]s for production of new [[virion]]s.
*''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 type]]s as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell.
==Applications==
===Basic research===
Viral vectors were originally developed as an alternative to [[transfection]] of [[naked DNA]] for [[molecular genetic]]s experiments. Compared to traditional methods such as [[calcium phosphate]] [[Precipitation (chemistry)|precipitation]], [[transduction (genetics)|transduction]] can ensure that nearly 100% of cells are infected without severely affecting cell viability.
Furthermore, some viruses [[retroviral integration|integrate]] into the cell [[genome]] facilitating stable expression. However, [[transfection]] is still the method of choice for many applications as construction of a viral vector is a much more laborious process.
[[Protein]] coding genes can be [[Gene expression|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.
Genes inserted and 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===
{{main|Gene therapy}}
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.<ref name="immunity">Beardsley T, ''February 2000'', [http://www.sciam.com/article.cfm?colID=20&articleID=000C73D5-BB64-1C75-9B81809EC588EF21| A tragic death clouds the future of an innovative treatment method.] ''Scientific American''</ref>
Some viral vectors, for instance lentiviruses, 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]] [[retrovirus|retroviral]] [[gene therapy]] trial conducted in 2002, two of the patients developed leukemia as a consequence of the treatment.<ref>McDowell N, ''15 January 2003'', [http://www.newscientist.com/article.ns?id=dn3271| New cancer case halts US gene therapy trials.] ''New Scientist''</ref> [[Viral vector#Adeno-associated viruses|Adeno-associated virus-based vectors]] are much safer in this respect as they always integrate at the same site in the human genome.
Vectors are also called vehicles.
===Vaccines===
Viruses expressing [[pathogen]] proteins are currently being developed as [[vaccine]]s against these pathogens, based on the same rationale as [[DNA vaccination|DNA vaccines]]. [[T-lymphocyte]]s recognize cells infected with [[intracellular]] [[parasite]]s based on the foreign proteins produced within the cell. [[T cell]] [[immunity (medical)|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 [[Albert Sabin|Sabin]] [[Polio vaccine]] and other [[attenuated]] vaccines. However, since viral vaccines contain only a small fraction of pathogen genes, they are much safer and sporadic infection by the pathogen is impossible. [[Viral vector#Adenoviruses|Adenoviruses]] are being actively developed as vaccines.
==Types of viral vectors==
===Retroviruses===
{{main|Retroviruses}}
[[Retrovirus]]es are the one of 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]] which allows integration into the host [[genome]]. They have been used in a number of FDA-approved clinical trials such as the [[SCID-X1]] trial.<!--
--><ref>{{cite journal | author= Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL, Bousso P, Deist FL, Fischer A. | title=Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. | journal= Science | year= 2000 | volume= 288 | issue= 5466 | pages= 669–72 | doi=10.1126/science.288.5466.669 | pmid=10784449}}</ref>
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 the their target cells and delivering their viral payload, but then fail to continue typical lytic pathway, which would typically result in cell lysis and death.
Conversely, replication-competent viral vectors contain all the necessary genes for virion synthesis, and will 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<ref>Principles of Retroviral Vector Design, http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=rv.section.4357</ref>. While this limits the introduction of many genomic sequences, most [[complementary DNA|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 (genetics)|transduction]]. As a result, cells such as [[neurons]] are very resistant to infection and transduction by retroviruses. There is a concern for [[insertional mutagenesis]] due to the integration into the host [[genome]] which can lead to [[cancer]] or [[leukemia]].
===Lentiviruses===
{{main|Lentivirus}}
[[Image:Lentiviral vector.png|thumb|right|300px|Packaging and transduction by a lentiviral vector.]]
[[Lentivirus]]es are a subclass of Retroviruses. They have recently been adapted as gene delivery vehicles (vectors) thanks to their ability to integrate into the [[genome]] of non-dividing cells, which is the unique feature of Lentiviruses as Retroviruses can infect only dividing cells. The viral genome in the form of [[RNA]] is [[reverse transcription|reverse-transcribed]] when the virus enters the cell to produce [[DNA]], which is then inserted into the genome at a random position 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 [[carcinogenesis|development]] of [[cancer]], which raises concerns for possible applications of lentiviruses in gene therapy.
For safety reasons lentiviral vectors never carry the genes required for their replication. To produce a lentivirus, several [[plasmid]]s are [[transfection|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 [[Transcription (genetics)|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.
===Adenoviruses===
{{main|Adenovirus}}
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 occasionally used in ''in vitro'' experiments. Their primary applications are in [[gene therapy]] and [[vaccination]]. Since humans commonly come in contact with [[adenoviruses]], which cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response with potentially dangerous consequences.<ref name="immunity"/> To overcome this problem scientists are currently investigating [[adenoviruses]] to which humans do not have immunity.
===Adeno-associated viruses===
{{main|Adeno-associated virus}}
===Nanoengineered Substances===
Substances such as [[Ormosil]] have been successfully used as a DNA vector.
==References==
<references />
==See also==
*[[Viral transformation]]
==External links==
*[http://www.natureprotocols.com/2007/02/14/production_of_neuronpreferenti.php Production of neuron-preferential lentiviral vectors (a protocol)]
*[http://www.natureprotocols.com/2007/01/24/placenta_specific_gene_manipul.php Placenta specific gene manipulation by transducing zona-free blastocyst using lentiviral vector (a protocol)]
* [http://www.genetherapynet.com Gene Therapy Net]
* [http://journals.cambridge.org/fulltext_content/ERM/ERM1_11/S1462399499000691sup002.htm A comparison of vectors in use for clinical gene transfer]
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