慢病毒原理

      慢病毒属于逆转录病毒科，为RNA病毒。慢病毒(Lentivirus)载体是以HIV-1(人类免疫缺陷I型病毒)为基础发展起来的基因治疗载体。它对分裂细胞和非分裂细胞均具有感染能力。该载体可以将外源基因有效地整合到宿主染色体上，从而达到持久性表达。可有效地感染神经元细胞、肝细胞、心肌细胞、肿瘤细胞、内皮细胞、干细胞等多种类型的细胞.

Lentiviral vectors can mediate the efficient delivery, integration and stable expression of transgenes in dividing as well as nondividing cells, either in vitro or in several organs in vivo. As such, they open exciting possibilities for both basic research and the genetic treatment of human diseases.

Lentivector particles are generated by co-expressing the virion packaging elements and the vector genome in a cell used as producer, for instance a 293 or 293T human embryonic kidney cell. In the case of 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, we designate the former elements as the LV packaging system the latter as the envelope.

In order to produce lentivectors you need 3 components:

  • vector, e.g. pWPTS, pWPXL, pWPI, pLVTHM
  • packaging system, e.g. pCMV-dR8.91, pCMV-dR8.74psPAX2 (2nd generation)
  • envelope plasmid, e.g. pMD2G

 

Three generations of HIV-based LV packaging systems have been successively 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, pol, responsible for the retrovirus-specific enzymes, and rev, which encodes a post-transcriptional regulator necessary for efficient gag and pol expression. a cDNA encoding rev 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 of the 3rd generation packaging combo: pMDL g/p RRE + pRSV-Rev.

 

All 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 lentivector with a chimeric 5'LTR e.g. CCL-, RRL-, etc, in which HIV promoter was 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 transient transfection of 293T cells with the second generation packaging system will satisfy 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.