Vectors Based on Lentiviruses
Retroviruses are broadly divided into two categories based on their genome complexity: simple and complex. The simple retroviruses contain only three genes, gag, pol, and env, while the complex retroviruses contain genes that code for a number of additional proteins that are responsible for regulating viral replication and interacting with the host cell immune response. In contrast with simple retroviruses, lentiviruses have evolved the ability to infect non-dividing cells, an attribute that significantly broadens the utility of lentiviral vectors to numerous target tissue and cell types. Human immunodeficiency virus (HIV) is a member of the lentiviradae genus also comprising simian immunodeficiency virus (SIV) and non-primate lentiviruses, such as equine infectious anemia virus (EIAV), caprine arthritis-encephalitis virus (CAEV) and the feline immunodeficiency viruses (FIV).
Lentiviral Vectors
The first lentiviral vectors developed were based on HIV-1. These were found to be efficient at transducing non-dividing cells and yet retained the ability of simple retrovirus vectors to integrate transgenes into the target cell genome, without triggering an inflammatory response. The design of replication-defective HIV-1 vectors is based on the strategy of segregation of the cis-acting elements in the HIV-1 genome from protein-encoding sequences. As an additional measure of safety, envelope-encoding sequences are usually separated from the rest of the HIV-1 packaging cassette. Based on this approach, the components required for the generation of HIV-1 based vectors are supplied in producer cells from three separate expression cassettes: envelope, packaging, and vector cassette.
Figure 1. Lentivirus and lentivirus vector.
- The Envelope Cassette
Substituting the parental HIV-1 envelope with the vesicular stomatitis virus G protein (VSV-G) was a breakthrough in lentiviral vector development. The VSV-G envelope confers three new features on lentiviral vector particles: (1) it stabilizes vector particles and therefore allows vector concentration by ultracentrifugation; (2) it mediates attachment of vector particles to phosphatidylserine molecules on target cells, thus dramatically broadening vector tropism; and (3) it directs lentivirus vector entry to an endocytic pathway, which reduces the requirements for viral accessory proteins for full infectivity. Several studies demonstrated that lentivirus vectors could be successfully pseudotyped with a variety of simple retrovirus envelope proteins. In general, titers of VSV-G pseudotyped lentivirus vectors are more than 10-fold higher than the typical titers of non-VSV-G pseudotyped lentivirus vectors.
- The Packaging Cassette
Excluding the envelope protein, the packaging cassette expresses all HIV-1 proteins required for vector particle production and efficient transduction of target cells. Unlike simple retroviruses, lentiviruses express a further six proteins in addition to the gag, pol, and env genes. These genes are termed accessory genes and include the vpu, vpr, vif, nef, and the virus regulatory proteins tat and rev, which control HIV-1 gene expression at transcriptional and posttranscriptional levels. The accessory proteins are essential for high-rate HIV-1 replication in vivo, and determine the pathogenic features of the virus. To improve the biosafety of the HIV-1 vectors, several packaging cassettes have been developed. The initial packaging construct, which was termed first-generation packaging cassette, contains all of the HIV-1 accessory genes. A later packaging construct, which is devoid of all of the accessory genes excluding the tat and the rev, is termed second-generation packaging cassette. Further deletion of the tat gene and separating the gag/pol and the rev genes into two expression cassettes resulted in the development of the third-generation packaging system.
- The Vector Cassette
The vector plasmid expresses the full-length vector RNA, containing all of the HIV-1 cis-acting elements and the transgene-expression cassette. The HIV-1 sequences in the vector included the 5' LTR and the 5' leader sequence, followed by 360 bp of the gag gene and 700 bp of the env gene. These sequences contain important cis-elements including the primer binding site (PBS), the splice donor site, the packaging signal, the RRE, and the splice-acceptor site. The internal promoter and the transgene sequences were located in the middle of the vector and were followed by 800 bp from the 3' end of the HIV-1 genome, which contains the 3' end of the nef gene, a polypurine tract (PPT) and the 3' LTR. To date, the common method of HIV-1 and other lentivirus vector production is based on transient transfection of three to four plasmids into 293T cells. In addition, several lentivirus vector-packaging cell lines have been developed as an alternative method of vector production.
Reference
- Cockrell, A. S.; et al. (2007). Gene delivery by lentivirus vectors. Molecular biotechnology. 36(3): 184-204.
