Lentivirus Preparation

Lentivirus Preparation

HIV-derived lentiviral vectors have been developed as a gene delivery system that can mediate the efficient delivery, integration, and sustained long-term expression of transgenes to dividing and non-dividing cells in vitro and in vivo. Over the years, lentiviral vectors have turned out to represent very powerful tools in basic and translational scientific research. As such, the facility has implemented the infrastructure and technology to produce high-titer lentiviral vectors. In addition, we have pursued their technological improvement for constitutive and conditional expression of cDNAs and shRNAs.

Lentivirus particles are generated by co-expressing the virion packaging elements and the vector genome 
via transient transfection in a producer cell line (e.g. HEK293T). For HIV-1-based vectors, the core and enzymatic components of 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. 
By convention, the former elements are referred to as the LV packaging system and the latter as the envelope.

3 components are required to produce an infectious lentivirus vector:
vector (e.g., pLU, pLKO, pGIPZ)
packaging system (e.g., pCMV-dR8.91, psPAX2 (2nd generation))
envelope plasmid, (e.g., pMD2G (VSVg))

Three generations of HIV-based LV packaging systems have been developed for production of lentivectors by transient transfection.

The first generation LV packaging system encompasses all HIV-1 genes besides the envelope.

The second generation LV packaging system is additionally deleted in all viral auxilliary genes, 
i.e., vpr, vif, vpu and nef. Examples: pCMV-dR8.91, pCMV-dR8.74, psPAX2

The third generation LV packaging system comprises only gag, coding for the virion main structural proteins and pol, responsible for the retrovirus-specific enzymes. 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.
Plasmid DNAs for the 3rd generation packaging system include pMDL g/p RRE and pRSV-Rev.

All lentiviral vectors that contain a wild-type 5'LTR (e.g., pGIPZ) need to be packaged with a 2nd generation packaging system, as wt 5'LTR requires TAT for maximal activation of transcription. The 3rd generation packaging system can only be used with a lentiviral vector with a chimeric 5'LTR e.g. CCL-, RRL-, etc, in which HIV promoter has been replaced with CMV or RSV, thus making them TAT-independent. The lentivectors carrying the chimeric 5'LTR can be packaged into both, 2nd or 3rd generation packaging system.

The production of vector particles by our facility uses a second generation packaging system, which satisfies most applications.

The 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 lose the transcriptional 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.