Review this way, the resulting provirus will be free of viral DNA responsible for viral replication and cell lysis. The resulting vector is replication dead and therefore nonmalignant and suitable for protein introduction (Markwoitz et al). The diagram below explains in detail the mechanism for production of retroviral vectors and the action of said vectors on target cells. suitable for protein introduction (Markwoitz et al). The diagram below explains in detail the mechanism for production of retroviral vectors and the action of said vectors on target cells. II. Advantages of Retroviral Vectors Defective Vector System A replication defective vector system refers to a retroviral vector without genes responsible for viral reproduction. This defect occurs naturally in certain mouse retroviruses because part of the normal viral genome has been replaced with a cDNA copy of a cellular oncogene. This type of vector is beneficial in gene therapy because its viral proteins do not have to be introduced to the host cell; rather, proteins can simply be provided in trans by the producer cell. No de novo protein synthesis is required in maintenance of the provirus, thus, immune response commonly associated with viral protein transfer and production is minimized. The repression of replication and reduced immune response associated with replication defective retroviruses makes them an attractive agent for protein and drug transfer (Hindmarsh et al). Well Documented Integration One of the primary advantages of retroviral vectors over others is that they are able to integrate their genetic information into the host cell efficiently, and the mechanism for this has been well documented. The process begins when the virus RNA genome is reverse transcribed into linear DNA before being converted into double stranded DNA. The ends of the long terminal repeats found at the termini of the linear viral DNA are recognized by integrase. The next step is formation of preintegration complexes, large protein structures composed of linear viral DNA; several viral proteins including matrix reverse transcriptase, nucleocapsid, and viral integrase; and at least two cellular proteins, high-mobility-group [HMG-I(Y)] and barrier to autointegration factor (BAF). This complex enters the nucleus via the nuclear pores or after the disintegration of the nuclear membrane in cell division. Once the complex enters the nucleus and associates with the chromosomes, viral integrase is released. This enzyme catalyzes the insertion of viral DNA into the host genome by bringing the linear viral DNA together with the host DNA. Lastly, a two base pair sequence is lost from each end of the viral DNA, four to seven base pairs are duplicated on the ends of the host DNA, and integrase binds the host and viral DNA. Cellular proteins mediate
Street Broad Scientific repair of damage to the newly generated provirus from the binding process. The excellent documentation and efficient insertion of desired genes into target cells make retroviruses ideal vectors for DNA transduction (Anson). Flexible Component Organization and Gene Expression Although in a rudimentary retroviral vector system the 5’ long terminating repeats acts as a promoter for the ordinarily coding for viral replication and virion maturation (Verma et al).The genes to be delivered by the vector are inserted or cloned into the genome construct and its expression is promoted by the 5’ long terminal repeat. The second component of the vector is a packaging cell line that delivers all the viral proteins coded for by the gag, pol, and env genes to the vector in trans. Proteins responsible for insertion of the vector genome into a host cell (transduction) are contained within the packaging construct and become active upon binding with the cell surface (Templeton).
III. Goals in Developing Retroviral Vectors Target Specificity One of the foremost goals in vector design is creating a vector that can target specific cells. Creating feature of a vector would grant it a significantly greater clinical relevance because it would transduce only in targeted cells. Development of this technology would allow in vivo delivery of a vector to an afflicted cell and would allow for treatment of specific cells using gene therapy. Proteins on the viral membrane surface mediate the mechanism by which a virus is guided towards its target cell in nature. The interaction of viral surface proteins with receptors on the cell surface detery mines entry of the virus into the cell. Thus, one step toward creating a target specific vector involves tagging the vector surface with the appropriate proteins to interact with the surface of the target cell. Upon binding of a vector to a cell surface receptor, the receptor will either cause a change in the physiological protein structure of the virus to grant it entry or it will cause acidification of the viral sheath to induce structural changes. This type of targeting is known as vector pseudotyping. Regulated Gene Expression Maintaining an appropriate level of gene expression in cells with transduced vector DNA has been problematic in retroviral vectors in the past. Integration of a vector genome into the provirus in the host cell is an advantage of current retroviral vectors, however, regulation of the gene expression of the vector DNA has not been effectively documented. This problem is true of all types of vectors; it is not limited to retroviruses in particular. There are two main barriers to effective gene regulation: interaction of the cell with vector promoter sequences and interaction of the host immune system with vector generated proteins. Although a promoter in the vector genome may drive gene Volume 1 | 2011-2012 | 31