Viral vector
As Viral vectors modified virus particles are specifically designated to be used in genetic engineering for it to inject genetic material into target cells. This can be a living organism (in vivo) or cells of a cell culture be (in vitro) cells.
The transport of DNA into a cell by a virus is referred to as transduction, the cells bearing such DNA, as transduced cells. Viral vectors used in basic research and gene therapy. Also, vaccines based on viral vectors are already in use. The technique of viral vectors was first used in the 1970s and since then constantly evolving. Viral vectors contain, in contrast to virus - like particles (VLP ) or liposomes with viral membrane proteins ( virosomes ) to a nucleic acid -making. This nucleic acid is then read either temporarily until it is removed or it is present as an episome in the cell nucleus before addition to the genome, or they will be permanently incorporated into the genome.
Naturally occurring viral vectors are for example the Polydnaviridae.
- 4.1 retroviruses 4.1.1 lentiviruses
- 4.1.2 Foamyviren
Properties of viral vectors
With a viral vector to introduce genetic material into target cells by transducing the desired DNA sequence must first be cloned into the genome of the virus. Certain DNA regions of the viral genome to be replaced in most cases, so that the viruses are not replication-competent because they lack such as regulatory sequences and the genes of enzymes, or capsid proteins. Such vectors can not reproduce themselves. Infectious particles can only be made if, for example, relevant information for the missing proteins and nucleic acids are provided separately by complementation in cell line that produces the vectors. Replication-deficient vectors have the advantage that they are in the target cell that does not contain this information, you can not multiply and are therefore used in preference to uncontrolled replication-competent vectors. By homologous recombination in the packaging cell line, however, replication-competent viruses can arise to some extent again. To avoid this, the genetic material is often changed so as part of the vector designs that they are replication in as many respects, eg by multiple deletions. If the genetic information on two different plasmids, is spoken by a two- plasmid system. To increase the safety of the vectors further, the genetic information on three or more different plasmids are distributed, then each must be co-transfected vector manufacture. In addition to the vector design, a protein design can take place. This allows the specific adaptation of various properties of the transgene and its expressed protein.
Requirements of a viral vector are:
- Safety and low genotoxicity
- Low cytotoxicity and symptoms
- Stability
- Cell type specificity
- Size of the genetic package capacitance
While viral vectors for gene therapy of genetic defects to generate a persistent gene expression, transient viral vectors are used for immunization against infectious diseases, for phage therapy of partially antibiotic-resistant bacteria for the treatment of tumors with oncolytic viruses or to generate induced pluripotent stem cells.
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 transfection may be ensured by the transduction that almost 100 % of the cells obtained, the genetic material without the viability of the cells is significantly reduced. In addition, some of the viruses and viral vectors derived from them will be able to integrate their DNA into the genome of the target cell so that expression of the DNA can be stably anchored, and may even be passed on to the daughter cells. Since the production of viral vectors is very complex, the steadily improving transfection, however, for many applications continue to be the better solution.
Genes that encode proteins can be expressed with the aid of viral vectors in order to examine the function of the protein. Vectors, especially retroviral vectors expressing stable marker genes such as GFP, can be used for the permanent labeling of cells in order to trace them and their descendants can. This happens for example in xenotransplantation.
In the vector of cloned genes encoding shRNAs and miRNAs, can be used for RNA interference experiments.
Gene Therapy
Several gene therapy trials have been conducted since the early 1990s, in which different viral vectors were used. With the help of these vectors is the defective gene in a monogenic hereditary disease, for example, the dysfunctional inherited gene can be replaced by a healthy one. Here, various difficulties depending on which vector and the method used arise. In vitro, significant progress has already been made, when used in the patients but these were always accompanied by risks, and therefore the gene therapy has not yet found wide application. The immune reactions of the patient to the vector may cause complications, thus in the case of Jesse Gelsinger which has been treated with an adenoviral vector 1999. For these reasons it will be necessary for each disease to be treated by gene therapy, to develop an appropriate and specific viral vector.
Retroviral vectors, for example, integrate their genomes into the genome of the host cell. The single integration is done at a random position in the genome, but overall as active genes in HIV or MLV promoter regions are strongly preferred, depending on type of virus specific locations. At this integration places the viral genome can interrupt genes of the host cell or naturally activate silenced genes, where one assumes that this happens more or less often depending on the integration patterns of the respective virus. This can lead to degeneration of the cell and thus to tumor formation, as happened in a gene therapy trial of Severe Combined Immunodeficiency ( SCID), which was carried out in France in 1999. Four of the 10 treated children developed leukemia as a result of treatment.
Vaccines
Transient viruses carrying DNA of pathogens for transient expression, are currently being tested as vaccines against pathogens. Bringing the viral vector DNA of the pathogen into the cell where it is expressed. The technique is thus an enhancement of all DNA vaccination.
History
Paul Berg 1976 used a modified SV40 virus, the DNA of bacteriophage lambda containing to infect cell culture kept in monkey kidney cells. Since the mid- 1980s, retroviral vectors are used which contain marker genes to highlight cells with and to pursue their development within an organism. Thereby making it possible to imagine, to use as a therapeutic genes into target cells.
Types of viral vectors
Retroviruses
Retroviruses are currently the focus of gene therapy research and due to their properties, the most widely used Vektoren.Rekombinante retroviruses can stably integrate into the host genome and are currently the only instrument to cure monogenic hereditary diseases permanently. Nearly 300 clinical studies have been carried out (2007) with retroviral vectors. The genus Gamma retrovirus associated retroviruses such as murine leukemia virus transduce mainly to exploiting dividend in division of cells and are therefore not suitable for transduction of resting cells such as nerve cells. Because gamma retroviral vectors carry the risk to be activated by the strong U3 enhancer elements in their LTRs potential Onkongene near its integration site, called SIN vectors have been developed ( by self inactivating ), which this enhancer in the 3'- LTR region are missing. After reverse transcription and integration of the provirus into the host genome, these also lack in the 5'- LTR region. It could be shown that the risk of Protoonkogenaktivierung thereby reduced to be completely prevented, it may not even with SIN vectors.
Lentiviruses
Lentiviruses are a genus within the retroviruses. You can, in contrast to gamma retroviruses infect quiescent cells and therefore have a potentially wider range of applications. The complex biology of lentiviruses is the reason why the development of vectors from lentiviruses is more difficult and therefore these vectors yielded useful results such as high titers only after a longer lead time. According to a published in November 2006, the first clinical trial in which lentiviral vectors were utilized in order to suppress the replication of HIV- 1 in patients with AIDS, positive balance was drawn. Also of lentiviral vectors SIN vectors were generated that mean a major step forward for safety.
Foamy viruses
Foamy viruses are a zoonotic subfamily within the retroviruses. An advantage over simple retroviruses example, represents the large genome of foamy viruses, so that even greater therapeutic genes can be packaged. In contrast to lentiviruses is mainly the general non-pathogenicity of foamy viruses emphasized so that is expected of a low hazard even when replication-competent vectors. Also, foamy viruses have more desirable properties, such as that in contrast to other retroviruses because of the genome dependent assembly of the virion and the Env dependent release not be unusable for gene therapy, virus-like particles formed, and in the integration of less than MLV or HIV active genes and transcription start sites are preferred. However, a problem arises, for example, the lack of foamy viruses Pseudotypisierbarkeit dar. Even with foamy viruses SIN vectors are used.
Adenoviruses
Unlike the lentiviruses, adenoviruses do not integrate into the genome of the cell and not be replicated during cell division. Their commitment to basic research is therefore limited. Their main field of application is the vaccination or of gene therapy, if only transient expression of the target gene is desired. This is the case in the fight against tumors with oncolytic viruses. In the third generation adenoviral vectors, the production of virions of cofactors in a packaging cell line dependent. Also AdV vectors induce a vector immunity, so you can use a serotype per patient only once.
Adeno -associated viruses
The adeno -associated virus (AAV ) in humans leads to asymptomatic coinfection during adenoviral infection. The adeno-associated viral vectors have a maximum length of eight DNA kilobases ( single-stranded ), which can still be packaged into virions. When using self - complementary DNA, the maximum length of a transgene is lowered to five kilobases in length about the virus titer is reduced. Because as with all viral vectors antibodies against the viral proteins (in this case against capsid proteins ) results from a congenital and an adaptive vector immunity, so that you can use to prevent premature degradation of the vector or excessive immune reactions serotype per patient only once, continuously develops new serotypes. However, integrations were found by AAV vectors in transcriptional active regions of the genome, which may contribute to the formation of tumors.
Baculoviruses
Baculoviral vectors are used in cell cultures of insect cells, inter alia, for the production of AAV vectors. The application in cell culture ( without adaptive immune system) allows continuous culture even in transient viruses. The use of insect cells reduces the risk of contamination with other pathogens of man that may infect insect cells in general. Insect cells also grow even at room temperature and without CO2 fumigation, but have partially different glycosylation patterns of their proteins.