Viral vector: Wikis

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Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes by a virus is termed transduction and the infected cells are described as transduced. Molecular biologists first harnessed this machinery in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the bacteriophage lambda to infect monkey kidney cells maintained in culture.[1]

Contents

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.

Applications

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Basic research

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, transduction 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. 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 expressed using viral vectors, commonly to study the function of the particular protein. Viral vectors, especially retroviruses, stably expressing marker genes 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

In the future gene therapy may provide a way to cure genetic disorders, such as severe combined immunodeficiency, cystic fibrosis or even Haemophilia A. Because these diseases result from mutations 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.[2]

Some viral vectors, for instance lentiviruses, insert their genomes at a seemingly random location on one of the host chromosomes, 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.[3] 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 vaccines against these pathogens, based on the same rationale as DNA vaccines. T-lymphocytes recognize cells infected with intracellular parasites 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 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. Adenoviruses are being actively developed as vaccines.

Types of viral vectors

Retroviruses

Retroviruses 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.[4]

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 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[5]. 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 a concern for insertional mutagenesis due to the integration into the host genome which can lead to cancer or leukemia.

Lentiviruses

Packaging and transduction by a lentiviral vector.

Lentiviruses 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 other Retroviruses can infect only dividing cells. The viral genome in the form of RNA is 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 oncogenes promoting the development of cancer, 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[6]. 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[7]. 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 plasmids 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 proteins, 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.

Adenoviruses

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.[2] To overcome this problem scientists are currently investigating adenoviruses to which humans do not have immunity.

Adeno-associated viruses

Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus 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. These features make AAV a very attractive candidate for creating viral vectors for gene therapy.[1]

Nanoengineered substances

Nonviral substances such as Ormosil have been used as DNA vectors and can deliver DNA loads to specifically targeted cells in living animals. (Ormosil stands for organically modified silica or silicate.)

See also

References

  1. ^ 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
  2. ^ a b Beardsley T, February 2000, A tragic death clouds the future of an innovative treatment method. Scientific American
  3. ^ McDowell N, 15 January 2003, New cancer case halts US gene therapy trials. New Scientist
  4. ^ 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. (2000). "Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.". Science 288 (5466): 669–72. doi:10.1126/science.288.5466.669. PMID 10784449.  
  5. ^ Principles of Retroviral Vector Design, http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=rv.section.4357
  6. ^ Cattoglio C, Facchini G, Sartori D, et al. Hot spots of retroviral integration in human CD34+ hematopoietic cells. Blood.2007 Sep 15;110(6):1770-1778
  7. ^ Montini E, Cesana D, Schmidt D, et al. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat Biotechnol. 2006 Jun;24(6):687-696.

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