Vectors Based on Gammaretroviruses
Retroviruses are a class of enveloped viruses that contain a single-stranded RNA molecule as the genome. Based upon common patterns of pathogenesis, retroviruses can be grouped into three basic subfamilies: oncogenic retroviruses, lentiviruses, and spumaviruses. The study of oncogenic retroviruses is of significant importance; it opened up the field of cellular growth control and it led to the discovery of proto-oncogenes, the mutation of which can cause cancer. Most oncogenic retroviruses (such as murine leukemia virus) belong to the simple retrovirus classification, while lentiviruses and spumaviruses (for example, human immunodeficiency virus) are complex retroviruses.
Retroviral Vectors
Retroviral vectors are derived from wild type retroviruses and are engineered to carry a foreign gene of interest into a target cell. Generally, the retroviral vectors are composed of the gene of interest and the cis-acting elements of the viral genome, with the removal of the trans-acting viral genes (e.g., gag, pol, and env).
For oncovirus-based vectors, such as the murine leukemia virus (MLV), the cis-acting elements refer to attachment sites (att), long terminal repeats (LTR), the primer binding site (PBS), the packaging signal C, and the polypurine tract (PPT) which are necessary for viral gene expression and replication.
For lentivirus-based vectors, such as human immunodeficiency virus (HIV-1), in addition to the above cis-acting elements, sequences that extend into the gag open reading frame are important for packaging. Therefore, HIV-1 vectors also contain the relevant portion of gag in which the translational initiation codon for gag itself has been mutated. HIV-1 based vectors also contain a portion of the env gene that includes the Rev response element (RRE).
Figure 1. The vector structures of MLV and HIV-1.
Once a retroviral vector has been constructed, there are several ways to express the genes of interest. Expression of the two genes can be achieved by the same LTR promoter if the second gene is expressed from a spliced message, or if the second gene can be translated using an internal ribosome entry site (IRES). The most common practice is for the gene of interest to be expressed by the LTR promoter and the endogenous gene expressed by an internal, heterologous promoter such as CMV, SV40, or a tissue-specific promoter. If the retroviral vector contains only one gene and it is expressed from the LTR promoter, the expression level is usually high. However, if the vector contains two genes that are expressed from different promoters, the level of expression of each gene is reduced.
Advantages
Retroviral vectors have a packaging size up to 9 kb, which is advantageous over adeno-associated viral vectors. Moreover, retroviral vectors have broad tropism. Once the vector genome integrates into the host cell genome, long-term transgene expression can be achieved; however, in some cells, there can be silencing unless insulator elements are included in the transgene expression cassette design. Preliminary studies suggest that retroviral vectors will be developed that drive the expression of short interfering RNAs (siRNA) in target cells, adding "knockdown" to the "knockout" functions of vectors. There is a need for robust insulating sequences that will enable positional independent expression from these vectors. In some applications, there is a need to insulate cellular genes from the effects of enhancer sequences in the vector. A further improvement for these applications would be to direct the site-specific integration of the virus into the genome.
Reference
- McTaggart, S.; et al. (2002). Retroviral vectors for human gene delivery. Biotechnology advances. 20(1): 1-31
