Viral vector
Encyclopedia
Viral vectors are a tool commonly used by molecular biologist
s to deliver genetic material into 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
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.
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 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 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.
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 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
retroviral
gene therapy
trial conducted in 2002, four of the patients developed leukemia as a consequence of the treatment. 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.
proteins are currently being developed as vaccine
s 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.
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 trial.
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
or leukemia
. 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.
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 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 oncogene
s 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. 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.
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. To overcome this problem scientists are currently investigating adenoviruses to which humans do not have immunity.
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
.)
Biologist
A biologist is a scientist devoted to and producing results in biology through the study of life. Typically biologists study organisms and their relationship to their environment. Biologists involved in basic research attempt to discover underlying mechanisms that govern how organisms work...
s to deliver genetic material into cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s. This process can be performed inside a living organism (in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
) or in cell culture
Cell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
(in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
). Virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es have evolved specialized molecular mechanisms to efficiently transport their genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
s inside the cells they infect. Delivery of gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s by a virus is termed transduction
Transduction (genetics)
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
and the infected cells are described as transduced. Molecular biologists first harnessed this machinery in the 1970s. Paul Berg
Paul Berg
Paul Berg is an American biochemist and professor emeritus at Stanford University. He was the recipient of the Nobel Prize in Chemistry in 1980, along with Walter Gilbert and Frederick Sanger. The award recognized their contributions to basic research involving nucleic acids...
used a modified SV40
SV40
SV40 is an abbreviation for Simian vacuolating virus 40 or Simian virus 40, a polyomavirus that is found in both monkeys and humans...
virus containing DNA from the bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
lambda
Lambda phage
Enterobacteria phage λ is a temperate bacteriophage that infects Escherichia coli.Lambda phage is a virus particle consisting of a head, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibers. The phage particle recognizes and binds to its host, E...
to infect monkey kidney
Kidney
The kidneys, organs with several functions, serve essential regulatory roles in most animals, including vertebrates and some invertebrates. They are essential in the urinary system and also serve homeostatic functions such as the regulation of electrolytes, maintenance of acid–base balance, and...
cells maintained in culture.
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 replicationViral replicationViral replication is the term used by virologists to describe the formation of biological viruses during the infection process in the target host cells. Viruses must first get into the cell before viral replication can occur. From the perspective of the virus, the purpose of viral replication is...
. Such a virus can efficiently infect cells but, once the infection has taken place, requires a helper virusHelper virusA helper virus is a virus used when producing copies of a helper dependent viral vector which does not have the ability to replicate on its own. The helper virus is used to coinfect cells alongside the viral vector and provides the necessary enzymes for replication of the genome of the viral vector....
to provide the missing proteinProteinProteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s for production of new virions. - Low toxicity: The viral vector should have a minimal effect on the physiologyPhysiologyPhysiology is the science of the function of living systems. This includes how organisms, organ systems, organs, cells, and bio-molecules carry out the chemical or physical functions that exist in a living system. The highest honor awarded in physiology is the Nobel Prize in Physiology or...
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 typeCell typeA cell type is a distinct morphological or functional form of cell. When a cell switches state from one cell type to another, it undergoes cellular differentiation. A list of distinct cell types in the adult human body may include several hundred distinct types.-References:...
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. Viruses modified in this manner are said to be pseudotypedPseudotypingPseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. The result is a pseudotyped virus particle.. With this method, the foreign viral envelope proteins can be used to alter host tropism or an increased/decreased stability of the...
. - 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.
Basic research
Viral vectors were originally developed as an alternative to transfectionTransfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
of naked DNA
Naked DNA
Naked DNA is histone-free DNA that is passed from cell to cell during a gene transfer process called transformation or transfection. In transformation , purified or naked DNA is taken up by the recipient cell which will give the recipient cell a new characteristic or phenotype...
for molecular genetics
Molecular genetics
Molecular genetics is the field of biology and genetics that studies the structure and function of genes at a molecular level. The field studies how the genes are transferred from generation to generation. Molecular genetics employs the methods of genetics and molecular biology...
experiments. Compared to traditional methods such as calcium phosphate
Calcium phosphate
Calcium phosphate is the name given to a family of minerals containing calcium ions together with orthophosphates , metaphosphates or pyrophosphates and occasionally hydrogen or hydroxide ions ....
precipitation
Precipitation (chemistry)
Precipitation is the formation of a solid in a solution or inside anothersolid during a chemical reaction or by diffusion in a solid. When the reaction occurs in a liquid, the solid formed is called the precipitate, or when compacted by a centrifuge, a pellet. The liquid remaining above the solid...
, transduction
Transduction (genetics)
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
can ensure that nearly 100% of cells are infected without severely affecting cell viability. Furthermore, some viruses integrate into the cell genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
facilitating stable expression.
However, transfection
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
is still the method of choice for many applications as construction of a viral vector is a much more laborious process.
Protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
coding genes can be expressed
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
using viral vectors, commonly to study the function of the particular protein. Viral vectors, especially retroviruses, stably expressing marker gene
Marker gene
A marker gene is a gene used in molecular biology to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA. There are two types of marker genes: a selectable marker and a marker for screening.-Selectable marker:...
s such as GFP
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
are widely used to permanently label cells to track them and their progeny, for example in xenotransplantation
Xenotransplantation
Xenotransplantation , is the transplantation of living cells, tissues or organs from one species to another. Such cells, tissues or organs are called xenografts or xenotransplants...
experiments, when cells infected in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
are implanted into a host animal.
Genes insertion is cheaper to carry out than gene knockout
Gene knockout
A gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...
. 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 therapyGene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...
may provide a way to cure genetic disorder
Genetic disorder
A genetic disorder is an illness caused by abnormalities in genes or chromosomes, especially a condition that is present from before birth. Most genetic disorders are quite rare and affect one person in every several thousands or millions....
s, such as severe combined immunodeficiency
Severe combined immunodeficiency
Severe combined immunodeficiency , is a genetic disorder in which both "arms" of the adaptive immune system are impaired due to a defect in one of several possible genes. SCID is a severe form of heritable immunodeficiency...
, cystic fibrosis
Cystic fibrosis
Cystic fibrosis is a recessive genetic disease affecting most critically the lungs, and also the pancreas, liver, and intestine...
or even Haemophilia A. Because these diseases result from mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
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
Jesse Gelsinger
Jesse Gelsinger was the first person publicly identified as having died in a clinical trial for gene therapy....
, who was treated using an adenoviral vector.
Some viral vectors, for instance lentiviruses, insert their genomes at a seemingly random location on one of the host chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
s, which can disturb the function of cellular genes and lead to cancer. In a severe combined immunodeficiency
Severe combined immunodeficiency
Severe combined immunodeficiency , is a genetic disorder in which both "arms" of the adaptive immune system are impaired due to a defect in one of several possible genes. SCID is a severe form of heritable immunodeficiency...
retroviral
Retrovirus
A retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...
gene therapy
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...
trial conducted in 2002, four of the patients developed leukemia as a consequence of the treatment. 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 pathogenPathogen
A pathogen gignomai "I give birth to") or infectious agent — colloquially, a germ — is a microbe or microorganism such as a virus, bacterium, prion, or fungus that causes disease in its animal or plant host...
proteins are currently being developed as vaccine
Vaccine
A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe or its toxins...
s against these pathogens, based on the same rationale as DNA vaccines
DNA vaccination
DNA vaccination is a technique for protecting an organism against disease by injecting it with genetically engineered DNA to produce an immunological response. Nucleic acid vaccines are still experimental, and have been applied to a number of viral, bacterial and parasitic models of disease, as...
. T-lymphocytes recognize cells infected with intracellular
Intracellular
Not to be confused with intercellular, meaning "between cells".In cell biology, molecular biology and related fields, the word intracellular means "inside the cell".It is used in contrast to extracellular...
parasites based on the foreign proteins produced within the cell. T cell
T cell
T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells , by the presence of a T cell receptor on the cell surface. They are...
immunity
Immunity (medical)
Immunity is a biological term that describes a state of having sufficient biological defenses to avoid infection, disease, or other unwanted biological invasion. Immunity involves both specific and non-specific components. The non-specific components act either as barriers or as eliminators of wide...
is crucial for protection against viral infections and such diseases as malaria
Malaria
Malaria is a mosquito-borne infectious disease of humans and other animals caused by eukaryotic protists of the genus Plasmodium. The disease results from the multiplication of Plasmodium parasites within red blood cells, causing symptoms that typically include fever and headache, in severe cases...
. A viral vaccine induces expression of pathogen proteins within host cells similarly to the Sabin
Albert Sabin
Albert Bruce Sabin was an American medical researcher best known for having developed an oral polio vaccine.-Life:...
Polio vaccine
Polio vaccine
Two polio vaccines are used throughout the world to combat poliomyelitis . The first was developed by Jonas Salk and first tested in 1952. Announced to the world by Salk on April 12, 1955, it consists of an injected dose of inactivated poliovirus. An oral vaccine was developed by Albert Sabin...
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.
Retroviruses
RetrovirusRetrovirus
A retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...
es are one of the mainstays of current gene therapy approaches. The recombinant retroviruses such as the Moloney murine leukemia virus
Murine leukemia virus
The murine leukemia viruses are retroviruses named for their ability to cause cancer in murine hosts. Some MLVs may infect other vertebrates. MLVs include both exogenous and endogenous viruses...
have the ability to integrate into the host genome in a stable fashion. They contain a reverse transcriptase
Reverse transcriptase
In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...
that allows integration into the host genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
. They have been used in a number of FDA-approved clinical trials such as the SCID-X1 trial.
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
Complementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
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
Transduction (genetics)
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
. As a result, cells such as neurons are very resistant to infection and transduction by retroviruses. There is concern that insertional mutagenesis
Insertional mutagenesis
Insertional mutagenesis is mutagenesis of DNA by the insertion of one or more bases.Insertional mutations can occur naturally, mediated by virus or transposon, or can be artificially created for research purposes in the lab.- Signature tagged mutagenesis :...
due to integration into the host genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
might lead to cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
or leukemia
Leukemia
Leukemia or leukaemia is a type of cancer of the blood or bone marrow characterized by an abnormal increase of immature white blood cells called "blasts". Leukemia is a broad term covering a spectrum of diseases...
. This concern remained theoretical until gene therapy for ten SCID-X1 patients using Maloney murine leukemia virus
Murine leukemia virus
The murine leukemia viruses are retroviruses named for their ability to cause cancer in murine hosts. Some MLVs may infect other vertebrates. MLVs include both exogenous and endogenous viruses...
resulted in two cases of leukemia caused by activation of the LMO2
LMO2
LIM domain only 2 , also known as LMO2, is a protein which in humans is encoded by the LMO2 gene.- Function :LMO2 encodes a cysteine-rich, two LIM domain protein that is required for yolk sac erythropoiesis. The LMO2 protein has a central and crucial role in hematopoietic development and is highly...
oncogene
Oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, they are often mutated or expressed at high levels.An oncogene is a gene found in the chromosomes of tumor cells whose activation is associated with the initial and continuing conversion of normal cells into cancer...
due to nearby integration of the vector.
Lentiviruses
Lentivirus
Lentivirus is a genus of slow viruses of the Retroviridae family, characterized by a long incubation period...
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
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
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
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
is reverse-transcribed when the virus enters the cell to produce DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, which is then inserted into the genome at a random position by the viral integrase
Integrase
Retroviral integrase is an enzyme produced by a retrovirus that enables its genetic material to be integrated into the DNA of the infected cell...
enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
. The vector, now called a provirus
Provirus
A provirus is a virus genome that is integrated into the DNA of a host cell.This state can be a stage of virus replication, or a state that persists over longer periods of time as either inactive viral infections or an endogenous retrovirus. In inactive viral infections the virus will not replicate...
, 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
Provirus
A provirus is a virus genome that is integrated into the DNA of a host cell.This state can be a stage of virus replication, or a state that persists over longer periods of time as either inactive viral infections or an endogenous retrovirus. In inactive viral infections the virus will not replicate...
can disturb the function of cellular genes and lead to activation of oncogene
Oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, they are often mutated or expressed at high levels.An oncogene is a gene found in the chromosomes of tumor cells whose activation is associated with the initial and continuing conversion of normal cells into cancer...
s promoting the development
Carcinogenesis
Carcinogenesis or oncogenesis is literally the creation of cancer. It is a process by which normal cells are transformed into cancer cells...
of cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
, 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
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
s are transfected
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
into a so-called packaging cell line, commonly HEK 293. One or more plasmids, generally referred to as packaging plasmids, encode the virion protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s, such as the capsid
Capsid
A capsid is the protein shell of a virus. It consists of several oligomeric structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or may not correspond to individual proteins, are called capsomeres. The capsid encloses the genetic...
and the reverse transcriptase
Reverse transcriptase
In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...
. Another plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
contains the genetic material to be delivered by the vector. It is transcribed
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
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 therapyGene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...
and vaccination
Vaccination
Vaccination is the administration of antigenic material to stimulate the immune system of an individual to develop adaptive immunity to a disease. Vaccines can prevent or ameliorate the effects of infection by many pathogens...
. 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. 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 that 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 OrmosilOrmosil
Ormosil is a shorthand phrase for organically modified silica or organically modified silicate. These are engineered materials that show great promise in a wide range of applications such as:...
have been used as DNA vectors and can deliver DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
loads to specifically targeted cells in living animals. (Ormosil stands for organically modified silica or silicate
Silicate
A silicate is a compound containing a silicon bearing anion. The great majority of silicates are oxides, but hexafluorosilicate and other anions are also included. This article focuses mainly on the Si-O anions. Silicates comprise the majority of the earth's crust, as well as the other...
.)