Virtually all lentiviral vectors are derived from the HIV-1 backbone - Lentivirus vector transduction
- Category: HIV based Lentiviral Vectors
- Created: Wednesday, 31 July 2019 21:49
- Written by Aline Do, Minh David, Sharon Parminder, Chahal Amine, A.Kamen
Lentivirus vectors are derived from the family Retroviridae, a family of enveloped, ssRNA viruses. Virtually all lentiviral vectors are derived from the HIV-1 backbone. The native tropism of HIV-1 is highly restrictive, and so the virus is commonly pseudotyped with Vesicular Stomatitis Virus G protein (VSV-G). VSV-G pseudotyping allows viral entry based on interaction with the LDL-receptor, expressed on all nucleated mammalian cells. Despite the broad tropism of VSV-G, lentivirus vector transduction is less efficient than adenoviral vectors.16 Various chemical and physical methods have been developed to improve transduction efficiency if necessary; Polybrene, a cationic polymer, is commonly added to cultures at concentrations ranging from 4 to 16 μg/mL to reduce charge repulsion between virions and the cell membrane and promote uptake. Alternatively, cell and lentivirus mixtures may be centrifuged together at a low speed (300–1600 g for 30–90 mins), a process known as “spinfection” or “spinnoculation”. Though the mechanistic underpinnings of the process are poorly understood, spinfection has been shown to increase transfection efficiency by roughly 5–10 fold.21
Given the lethal nature of the HIV-1 virus from which these vectors are derived, successive generations of lentiviral vector have chiefly sought to improve vector safety.
Third generation lentiviral vectors currently in use encode no viral proteins, only a few cis-acting sequence elements required for packaging, reverse transcription, and efficient expression. During vector production, essential viral proteins are supplied in trans on multiple plasmids to reduce the possibility of successful recombination, while virulence factors nonessential for in vitro replication are omitted entirely.22 Newer self-inactivating vectors also improve safety by making alterations to the 3′ and 5′ long terminal repeats of the virus, preventing the transcription of full-length viral RNA after integration and allowing the tat transcriptional regulator to be removed, respectively. It has been speculated that accidental exposure to lentiviral vectors could result in insertional mutagenesis, a fear found on observed tendency for the closely related γ-retroviral vectors to integrate in proximity to proto-oncogenes. Extensive analysis has shown though that lentiviral vectors have a very different preference for insertion sites, and so hazardous insertional mutagenesis is unlikely.23 As a result of these incremental improvements in vector design, lentiviral vectors are generally regarded as quite safe, and in some cases are handled at biosafety level 1, though more commonly at level 2.
The deletion of all viral proteins allows the lentiviral backbone to accommodate transgenes of up to 8.5 kb in size. Unlike adenoviruses, the backbone of lentiviral vectors is reverse transcribed and stably integrated into the host cell genome. While the requirement for prior reverse transcription and integration means that transgene expression is delayed to 24–48 h posttransduction (compared to <12 h for adenovirus vectors), this facilitates stable and heritable transgene expression in transduced cells. For this reason, lentiviruses have seen extensive use in the generation of stable cell lines, allowing integration of expression cassettes at efficiencies orders of magnitude higher than random plasmid integration or homologous recombination techniques would allow. To discourage promotor methylation and other epigenetic modifications that would silence transgene expression over time, selectable markers (either fluorescence or drug selection) are commonly incorporated onto the vector backbone.
Source : https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/lentivirus-vector