Lentiviral vectors derived from the human immunodeficiency virus (HIV-1) have become major tools for gene delivery in mammalian cells. The advantageous feature of lentivirus vectors is the ability to mediate potent transduction and stable expression into dividing and non-dividing cells both in vitro and in vivo.
The HIV-1 genome contains 9 open reading frames encoding at least 15 distinct proteins involved in the infectious cycle, including structural and regulatory proteins. In addition, there are a number of cis-acting elements required at various stages of the viral life cycle. These include the long terminal repeats (LTRs), the TAT activation region (TAR), splice donor and acceptor sites, packaging and dimerization signal (Ψ), Rev-responsive element (RRE) and the central and terminal polypurine tracts (PPT).
The earliest lentiviral vectors were replication-competent viruses carrying transgenes.To make these vectors safer, HIV vectors have evolved througha series of modifications to separate viral sequences needed for packaging and production from those encoding viral proteins.
First-generation HIV-1-based lentiviral vectors splited vector components into three plasmids to increasing safety: (i) a packaging construct; (ii) an Env plasmid encoding a viral glycoprotein; and (iii) a transfer vector genome construct. The packaging and enveloping plasmids have been specifically engineered without either a packaging signal or LTRs to avoid their transmission into vector particles and to reduce the production of RCLs(replicationcompetent lentiviruses) in vector preparations.
Second-generation vectors have been developed by modifying accessory genes proteins (Vif, Vpu, Vpr or Nef) in the system.These accessory proteins can be deleted without affecting viral replication in certain human lymphoid cell lines.
Third-generation lentiviral vector system consists of four plasmids.The packaging vector is split into two plasmids: one encoding Rev and one encoding Gag and Pol.Tat-independence is achieved by replacing the U3 promoter region of the 5'-LTR in the transfer vector with strong viral promoters from CMV or RSV [3,4]. The third-generation system was effectively reduced the production of RCLs and increased biosafety.
Sino Biological's HIV-based lentiviral vector systems are designed to maximize their biosafety features:
1) Deleting the U3 region from 5'LTR and replaced with a strong viral promoter CMV in the vector plasmid result in Tat-independent transcription but still maintaining high levels of expression.
2) A deletion in the U3 region of 3'LTR ensures self-inactivation of the lentiviral construct after transduction and integration into genomic DNA of the target cells.
3) The number of lentiviral genes necessary for packaging, replication and transduction is reduced to three (gag, pol, rev). In the third-generation lentiviral vector system gag-pro and pol are further separated onto two plasmids,and the packaging plasmids share no significant homology to any of the expression lentivectors.These measures greatly prevent generation of RCLs.
4) Compared with Mulv retroviral vector, the lentiviral vector prefer to integrated into active transcription regions of the host genome,which activated the silence of the original cancer gene risk was lower than Mulv retroviral vector.
Before vector infection of target cells, titration of vectors produced is required to adjust vector doses. Vector titres are also critical to evaluate transduction efficieincy. Titration can be accomplished using different methods:(1) measuring the quantity of vector particle components (physical particle numbers/p24 capsid concentration/RT activity/genome copy numbers) in vector preparations; (2) measuring proviral vector DNA copy numbers in infected cells; or (3) measuring transgenes expressed in cells (by flow cytometry or immunostaining).
The p24 ELISA methods specifically measure the amount of p24 capsid protein present in the viral supernatant and then correlates the level of p24 to virus titer. Whereas the p24 concentration measures both functional and non-functional vector particles,virus titer will be overestimated. Moreover,titration with viral genomic RNA copy numbers and p24 concentrations has been shown to be rather poor in predicting vector transduction efficiency.
In contrast,detecting proviral DNA copy numbers in infected cells by real-time PCR can evaluate infectious vector copy numbers.This method is suitable if the vector has no marker gene or the transgene is driven by a certain tissue-specific promoter.For vectors that contain a selectable marker, cells are infected with serial dilutions of the virus stock and then selected for stable transductants using the appropriate antibiotic. Titers are calculated from the number of drug-resistant colonies that develop after selection is completed. For vectors containing a fluorescent marker, cells can be transduced and counted using fluorescence and flow cytometry. Titers determined in this manner are generally higher than those determined by antibiotic selection.
1, Trono, D. Lentiviral Vectors (Berlin-Heidelberg, Springer-Verlag, 2002).
2, Sakuma, T., Barry, M. A. and Ikeda, Y. (2012) Lentiviral vectors: basic to translational. J. Biochem. 443, 603–618
3, Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D. and Naldini, L.(1998) A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471
4, Kim, V. N., Mitrophanous, K., Kingsman, S. M. and Kingsman, A. J. (1998) Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. J.Virol. 72, 811–816
5, Geraerts, M., Willems, S., Baekelandt, V., Debyser, Z. and Gijsbers, R. (2006) Comparison of lentiviral vector titration methods. BMC Biotechnol. 6, 34