Viral vectors are a tool commonly used by biologists to deliver genetic material into cells inside a living organism or cultured in vitro. Viruses have evolved specialised 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] A biologist is a scientist devoted to and producing results in biology through the study of organisms. ... Genetic material is the material used to store genetic information for a living organism. ... A cell is a single unit or compartment, enclosed by a border or wall. ... Epithelial cells in culture, stained for keratin (red) and DNA (green) Cell culture is the term applied when cells are grown in a synthetic environment. ... Wiktionary has a definition of: In vitro In vitro (Latin: within glass) means within a test tube, or, more generally, outside a living organism or cell. ... The human immunodeficiency virus (HIV) A bacteriophage virus A virus is a submicroscopic parasitic particle that infects cells in biological organisms. ... In biology the genome of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). ... This stylistic schematic diagram shows a gene in relation to the double helix structure of DNA and to a chromosome (right). ... The word transduction has several meanings: In developmental psychology, transduction is reasoning from specific cases to specific cases, typically employed by children. ... Molecular biology is the study of biology at a molecular level. ... Paul Berg, born June 30, 1926 in Brooklyn, New York, USA, is an American biochemist and professor emeritus at Stanford University. ... SV40 is an abbreviation for Simian vacuolating virus 40 or Simian virus 40, a polyomavirus that is found in both monkeys and humans. ... A phage (also called bacteriophage) (in Greek phageton = food/consumption) is a small virus that infects only bacteria. ... Enterobacteria phage λ (lambda phage) is a temperate bacteriophage that infects Escherichia coli. ... Human kidneys viewed from behind with spine removed The kidneys are bean-shaped excretory organs in vertebrates. ...

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.

A pathogen (literally birth of pain from the Greek παθογένεια) is a biological agent that can cause disease to its host. ... Viral replication is the term used by virologists to describe the propagation of biological viruses during the infection process in the target host cells. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... A common alternate meaning of virus is computer virus. ... Physiology (in Greek physis = nature and logos = word) is the study of the mechanical, physical, and biochemical functions of living organisms. ... A cell type is a distinct morphological or functional form of cell. ...

Applications

Basic research

Viral vectors were originally developed as an alternative to transfection of naked DNA for molecular genetic 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 into the vector can encode shRNAs and miRNAs used to efficiently block or silence production of a specific protein. Such knock-down experiments are much quicker 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. Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. ... Naked DNA is histone-free DNA. Research on the use of naked DNA for vaccines and gene therapy has shown some initial success, but have not yet resulted in any generally available therapy. ... Molecular genetics is the field of biology which studies the structure and function of genes at a molecular level. ... Calcium phosphate is the name given to a family of minerals containing calcium ions (Ca2+) together with orthophosphates (PO43-), metaphosphates or pyrophosphates (P2O74-) and occasionally hydrogen or hydroxide ions. ... The word transduction has several meanings: In developmental psychology, transduction is reasoning from specific cases to specific cases, typically employed by children. ... In biology the genome of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). ... Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... Gene expression, also called protein expression or often simply expression is the process by which a genes DNA sequence is converted into the structures and functions of a cell. ... A marker gene is used in molecular biology to determine if a piece of DNA has been successfully inserted into the host organism. ... The initialism GFP may refer to Green fluorescent protein, a fluorescent marker frequently used in biology. ... Xenotransplantation is the transplantation of cells, tissues or organs from one species to another such as from pigs to humans. ... Wiktionary has a definition of: In vitro In vitro (Latin: within glass) means within a test tube, or, more generally, outside a living organism or cell. ... ... In genetics, a miRNA (micro-RNA) is a form of single-stranded RNA which is typically 20-25 nucleotides long, and is thought to regulate the expression of other genes. ... In molecular biology, RNA interference (RNAi) is a mechanism in which the presence of small fragments of double-stranded RNA (dsRNA) whose sequence matches a given gene interferes with the expression of that gene. ... A gene knockout is a genetically engineered organism that carries one or more genes in its chromosomes that has been made inoperative. ...

Gene therapy

Main article: Gene therapy

In the future gene therapy may provide a way to cure genetic disorders, such as severe combined immunodeficiency or cystic fibrosis. Several gene therapy trials have used viruses to deliver 'good' genes to the cells of the patient's body. Gene therapy using an Adenovirus vector. ... Gene therapy using an Adenovirus vector. ... A genetic disorder, or genetic disease is a disease caused by abnormal expression of one or more genes in a person causing a clinical phenotype. ... Severe Combined Immunodeficiency, or SCID, is a genetic disorder in which both arms (B cells and T cells) of the adaptive immune system are crippled, due to a defect in one of several possible genes. ...

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 lead to the death of Jesse Gelsinger, who was treated using an adenoviral vector.^[2] A request has been made on Wikipedia for this article to be deleted in accordance with the deletion policy. ... Jesse Gelsinger was the first patient ever to die in clinical trials for gene therapy. ...

Some viral vectors, for instance lentiviruses, insert their genomes at a 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, two 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. Figure 1: Chromosome. ... Severe Combined Immunodeficiency, or SCID, is a genetic disorder in which both arms (B cells and T cells) of the adaptive immune system are crippled, due to a defect in one of several possible genes. ... Genera Alpharetrovirus Betaretrovirus Gammaretrovirus Deltaretrovirus Epsilonretrovirus Lentivirus Spumavirus A retrovirus is a virus which has a genome consisting of two RNA molecules, which may or may not be identical. ... Gene therapy using an Adenovirus vector. ...

Vaccines

Viruses expressing pathogen proteins are currently being developed as vaccines against these pathogens, based on the same rationale as DNA vaccines. T-lymphocytes recognise 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. A pathogen or infectious agent is a biological agent that causes disease or illness to its host. ... A vaccine is an antigenic preparation used to produce active immunity to a disease, in order to prevent or ameliorate the effects of infection by any natural or wild strain of the organism. ... DNA vaccination is a proposed experimental technique for protecting an organism against disease by injecting it with naked DNA to produce an immunological response. ... T cells are a subset of lymphocytes that play a large role in the immune response. ... In cell biology, molecular biology and related fields, the word intracellular means inside the cell. It is used in contrast to extracellular (outside the cell). ... A parasite is an organism that spends a significant portion of its life in or on the living tissue of a host organism and which causes harm to the host without immediately killing it. ... T cells are a subset of lymphocytes that play a large role in the immune response. ... In a medical sense, immunity is a state of having sufficient biological defenses to avoid infection, disease, or other unwanted biological invasion. ... Red blood cell infected with Malaria, derived from mala aria (Medieval Italian for bad air) and formerly called ague or marsh fever in English, is an infectious disease which causes about 350-500 million infections with humans and approximately 1. ... This article or section is missing references or citation of sources. ... Two polio vaccines are used throughout the world to combat polio. ... Attenuation is the decrease of the amount, force, magnitude, or value of something. ...

Types of viral vectors

Lentiviruses

Main article: Lentivirus

Lentiviruses are widely adapted as vectors thanks to their ability integrate into the genome of non-dividing as well as 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. // Comments A lentivirus is a genus of slow viruses of the retroviridae family, characterized by long incubation period. ... // Comments A lentivirus is a genus of slow viruses of the retroviridae family, characterized by long incubation period. ... In biology the genome of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). ... Ribonucleic acid (RNA) is a nucleic acid polymer consisting of covalently bound nucleotides. ... Reverse transcriptase is an enzyme used by all retroviruses and retrotransposons that transcribes the genetic information from the virus or retrotransposon from RNA into DNA, which can integrate into the host genome. ... The general structure of a section of DNA Deoxyribonucleic acid (DNA) is a nucleic acid —usually in the form of a double helix— that contains the genetic instructions specifying the biological development of all cellular forms of life, and most viruses. ... Integrase is a protein produced by a virus that enables genetic material that is helpful to the virus, proviral DNA, to be integrated into the DNA of the infected cell. ... Ribbon diagram of the enzyme TIM. TIM is catalytically perfect, meaning its conversion rate is limited, or nearly limited to its substrate diffusion rate. ... A provirus is a retrovirus that has integrated itself into the DNA of a host cell. ... A provirus is a retrovirus that has integrated itself into the DNA of a host cell. ... An oncogene is a modified gene that increases the malignancy of a tumor cell. ... Cancers are caused by a series of mutations. ... When normal cells are damaged beyond repair, they are eliminated by apoptosis. ...

Production of lentiviral vectors

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. Figure 1: Schematic drawing of a bacterium with plasmids enclosed. ... Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. ... Epithelial cells in culture, stained for keratin (red) and DNA (green) Cell culture is the term applied when growing cells in a synthetic environment. ... A common alternate meaning of virus is computer virus. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... The outer shell of a virus is called the capsid. ... To meet Wikipedias quality standards, this article or section may require cleanup. ... Transcription may be one of the following: In linguistics, transcription is the conversion of spoken words into written language. ...

Adenoviruses

Main article: 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 reasearch, 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 that humans do not have immunity to. Genera Mastadenovirus Aviadenovirus Atadenovirus Siadenovirus Adenoviruses are viruses of the family Adenoviridae. ... Gene therapy using an Adenovirus vector. ... Vaccination is the process of administering live, albeit weakened, microbes to patients, with the intent of conferring immunity against a targeted form of a related disease agent. ...

Adeno-associated viruses

Main article: Adeno-associated virus

Adeno associated virus is a virus associated with Adenovirus. ...

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