Lentiviral Vectors

Lentiviruses (lenti- is slow in Latin) are genus of slow viruses of the Retrovidae family, characterized by a long incubation period. Lentiviruses, including the HIV (Human), SIV (simian,) FIV (feline), are all pathogenic to humans. Lentiviruses can deliver significant amount of genetic information into the DNA of the host cell, and can target both dividing and non-dividing cells (unlike Retroviruses that infect only dividing cells). The native envelop of Lentivirus based vectors (the HIV-1) contains the Gp120 glycoprotein which is recognized by the CD4 receptor on the target cells (Lymphocytes). In order to broaden the range of target cells the native envelop was replaced (pseudotyped) by vesicular stomatitis virus G (VSVG).
All these unique features render Lentiviruses as the most efficient method for gene delivery. They are used to replace a damaged/missing gene in gene therapy, and in basic science are used to introduce or silence genes in a wide verity of models. It is an excellent method replacing non-efficient transfections, used for in vivo labeling, transgenic mice, high throughput screening and more.

The attractive features of Lentiviruses, especially with HIV-1 based Lentivirus vectors, raises Biosafety issues. Laboratory workers handling Lentivirus are the risk group. The biosafety level is enhanced BL2 (BL2+).

Lentivirus penetration may occur

  • Through the skin - via puncture or absorption (scratches, cuts, dermatitis, or other lesions)
  • Through mucous membrane - eyes, nose, and mouth.

Strict compliance to the following guidelines will prevent exposure

The basic principle is to avoid contamination with the recombinant virus, through droplets/aerosol/ skin puncture, therefore:

  1. No glass or sharps are allowed (use plastic pipets, blunt end needle).
  2. Centrifuge only in biohazard centrifuge.
  3. Use only vacuum aided filtration units (contact the Biosafety Officer for details).
  4. All the work should be contained within a biological hood.

According to the decision of the Institutional Biological safety Committee, Lentivirus work will be carried out in a dedicated departmental room, or in the tissue culture room of a group (pending the approval of a biosafety officer) and is under the responsibility of the principal investigator. Any work in this room should comply with the guidelines above, even when not working with lenti vectors.

We recommend using third generation plasmids, which allow production of replication-deficient viruses, such as Invitrogen Virapower system (recommended by the NIH). This system incorporates several safety features as follows:

  1. Tat, which is critical for activating viral transcription, is not expressed in the system.
  2. The vector is “self-inactivating” due to a deletion in the 3' LTR (U3).
  3. The genes encoding essential structural and functional proteins are not included in the lenti vector and are temporarily added via 3 separate plasmids (gag-pol, rev and VSV-G), thus reducing the chance of creating a replication competent virion.

When working with second generation lenti systems (a total of 3 plasmids), each virion production is a separate statistical event and therefore each prep should be tested for replication-competent lentiviruses (contact a Biosafety Officer for details).