A LENTIVIRAL VECTOR AS A POWERFUL TOOL
Lentiviral vectors can mediate the efficient delivery, integration and stable or controlled expression of transgenes or shRNAs in dividing as well as nondividing cells, either in vitro or in several organs in vivo. As such, it opens exciting possibilities for both basic research and the genetic treatment of human diseases.
HOW TO PRODUCE LENTIVECTOR PARTICLES
Lentivector particles are generated by co-transfection of plasmids coding for the virion packaging system (LV packaging), the vector genome carrying your gene of interest (LV vector) and the plasmid coding for an envelope, in a cell used as producer, for instance 293T (human embryonic kidney cell).
In the case of HIV-1 based vectors, the structural and enzymatic components ot the virion come from HIV-1, while the envelope is derived from a heterologous virus, most often vesicular stomatitis virus (VSV) due to the high stability and broad tropism of its G protein.
Three generations of HIV-based LV packaging systems have been successively developed for production of lentivectors by transient transfections :
First generation : LV packaging system encompasses all HIV-1 genes except the envelope
Second generation: will fit most of the experiments. LV packaging system is additionally deleted for all viral auxilliary genes, i.e. vpr, vif, vpu and nef (example: pCMV-dR8.91, pCMV-dR8.74, psPAX2)
Third generation : LV packaging system comprises only gag, coding for the virion main sturctural proteins and pol, coding for the retrovirus-specific enymes. A cDNA encoding rev, which encodes a post-transcriptional regulator necessary for efficient gag and pol expression, is provided on a separate plasmid. The third generation packaging system offers maximal biosafety but is more cumbersome, involving the transfection of four different plasmids in the producer cells. (example : pMDL g/p RRE + pRSV-Rev + pMD2G + pLV vector)
Most of our lentivectors contain wt 5’LTR and can be packaged only using 2nd generation packaging system (as wt 5′ LTR requires TAT for activation).
If you wish to use 3rd generation packaging system you need to have a LV vector with a chimeric 5’LTR e.g CCL-, RRL-, etc, in which HIV promoter was replaced with a CMV or RSV promoter, thus making them TAT-independent. The LV vectors carrying the chimeric 5’LTR can be packaged into both, 2nd and 3rd LVgeneration packaging system.
The production of vector particles by transient transfection of 293T cells with the second generation packaging system will satisfy most applications.
The LV vector itself is the only genetic material transferred to the target cells. It typically comprises the transgene cassette flanked by cis-acting elements necessary for its encapsidation, reverse transcription and integration.
As previously done with oncoretroviral vectors, advantage was taken of the gymnastics of reverse transcription to engineer self-inactivating (SIN) HIV-1-derived vectors, which loose the transcripitional capacity of the viral long terminal repeat (LTR) once transferred to target cells. This minimizes the risk of emergence of replication competent recombinants (RCR) and avoids problems linked to promoter interference.
The first-generation lentiviral vectors were manufactured using a packaging system that comprised all HIV genes but the envelope (Naldini et al., 1996).
In a so-called second-generation system, five of the nine HIV-1 genes were eliminated, leaving the gag and pol reading frames, which encode for the structural and enzymatic components of the virion, respectively, and the tat and rev genes, fulfilling transcriptional and post-transcriptional functions (Zufferey et al., 1997). Sensitive tests have so far failed to detect replication-competent-recombinants (RCRs) with this system. This good safety record, combined with its high efficiency and ease of use, explains why the second-generation lentiviral vector packaging system is utilized for most experimental purposes.
In a third-generation system, geared up towards clinical applications, only gag, pol, and rev genes are still present, using a chimeric 5′ LTR (long terminal repeat) to ensure transcription in the absence of Tat.
The genetic information contained in the vector genome is the only one transferred to the target cells.
Early genomic vectors were composed of the following components: the 5′ LTR, the major splice donor, the packaging signal (encompassing the 5′ part of the gag gene), the Rev responsive element (RRE), the envelope splice acceptor, the internal expression cassette containing the transgene, and the 3′ LTR .
In the latest generations, several improvements have been introduced.
The Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) has been added to increase the overall levels of transcripts both in producer and target cells, hence increasing titers and transgene expression (Zufferey et al., 1999).
The central polypurine tract of HIV has also been added back in the central portion of the genome of the transgene RNA (Follenzi et al., 2000; Zennou et al., 2000). This increases titers at least in some targets.
The U3 region 3′ LTR is essential for the replication of a wild-type retrovirus, since it contains the viral promoter in its RNA genome. It is dispensable for a replication-defective vector and has been deleted to remove all transcriptionally active sequences, creating the so-called selfinactivating (SIN) LTR (Zufferey et al., 1998). SIN vectors are thus unable to reconstitute their promoter and are safer than their counterparts with full-length LTRs. Finally, chimeric 5′ LTRs have been constructed, in order to render the LV promoter Tat-independent. This has been achieved by replacing the U3 region of the 5′ LTR with either the CMV enhancer (CCL LTR) or the corresponding Rous sarcoma virus (RSV) U3 sequence (RRL LTR; Dull et al., 1998). Vectors containing such promoters can be produced at high titers in the absence of the Tat HIV transactivator.
The latest generation with a chimeric LTR that can be produced in the absence of Tat (also called third generation) represents the system of choice for future therapeutics projects. In the laboratory, however, this third generation is not mandatory, and the second-generation system offers a high level of safety for P2 conditions. The second-generation packaging system also has the advantage of working on both second- and third-generation vectors. Thus, for in vitro and vivo research, it is easier to work with one all-purpose packaging plasmid, such as the pCMVR8.74, which encodes for the HIV-1 Gag, Gag/Pol, Tat, and Rev proteins.
Evolution in the design of transfer and packaging plasmids for hiv-1 based lv vectors
System used in the facility