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).

 

Biosafety guidelines and equipment requirements

  1. Obligatory equipment in a lenti room:
    • Biological hood (Class II)
    • Biohazard Centrifuge
    • Microscope
    • Incubator
    • Biosafety waste disposal equipment
    • A set of micro pipettes
    • Spill kit (face shield and shoe covers)
  2. Entrance to the room:
    1. Restricted entrance for authorized personnel only.
    2. Signs should be posted on the entrance stating the use of Lentivirus (obtain at the safety unit).

 

  1. Before starting your work:
    1. Prepare decontamination solutions (as specified below).
    2. Prepare a biohazard bag for solid waste inside the biological hood and another biohazard bag in the biohazard bucket.
    3. Wear personal protective equipment: a disposal laboratory-coat, 2 pairs of gloves and safety glasses. The outer gloves should be placed on top of the coat sleeve to protect the wrist. A water resistant disposable sleeve can be used on top of the lab coat sleeve.

 

Wear a surgical face mask when working with the virus (collection/ filtration/ centrifugation/ infecting cells) and at risk from mucous membrane exposure by contaminated hands.

 

When performing procedures that produce aerosols outside the biological hood, contact the safety unit for a risk assessment. Additional protective equipment may be needed.

 

  1. Working with lenti vectors:
    1. Shut the T.C door while working with Lentivirus (collection and infection).
    2. Use tips and pipettes containing filter.
    3. No sharps/glass are allowed.
    4. Disposable equipment (pipettes, flasks/plates) must be decontaminated before discarding it to the biohazard bag (details under decontamination).
    5. No other vectors are allowed in biological hood during the work with Lentivirus production.
    6. Do not leave virus-containing solutions unattended in the hood or in the centrifuge.
    7. You must not leave the Lentivirus approved room to any other room wearing the disposable lab-coat. You may leave the disposable lab-coat in the lenti room. At the end of work all disposable personal protective equipment (gloves and disposable lab-coat) should be discarded in a new biohazard bag located inside the approved room.
    8. Cells for virus packaging and for infection should preferably be grown in flasks (not in plates) and placed in the incubator on a tray (to contain spillage). In case the incubator is also used for growing non-infected cells, the infected cells should be placed at the bottom shelf.
    9. Do not touch anything outside the hood with contaminated gloves. Replace outer gloves upon leaving the biological hood (dispose contaminated gloves in the biohazard bag inside the hood).
    10. Centrifugation must be carried out in a biohazard centrifuge or in aerosol sealed tubes.
    11. For using an ultracentrifuge (located in another room), follow these instructions:
  • Post a clear sign notifying the use of Lentivirus, as well as information specifying the length of your run, your name and group and your mobile phone number.
  • Balance the plastic tubes by adding an exact volume of medium containing virus inside the biological hood of the approved room.
  • When filling, do not exceed 75% of the tubes volume.
  • Gently insert the plastic tube into the metal bucket, paying attention to avoid splashes.
  • Cover the metal bucket using the provided metal screw and O-ring.
  • Spray the exterior of the metal bucket and covers with 70% Ethanol, change your glove and the rest of your personal disposable protection. You may leave the room wearing a clean fabric lab-coat to access departmental ultracentrifuge room.
  • Take the sealed tubes to the centrifuge using the provided stand and place them inside a carrier such as a cooler.
  • After centrifugation open the lentivirus containing tubes only inside the biological hood in the Lentivirus approved room.
  • Clean the metal centrifuge buckets and covers (inside and outside), the ultracentrifuge and the rotor using 70% Ethanol or Bactwipe, even if you did not notice a spill.

 

 

    1. Wash the cells four times with media in order to work outside the lenti room, for example: using an instrument or working on the bench (FACS, microscopy, harvesting cells). Washing the cells reduces lenti titer to below the infective dose and thus it requires only BL2 practices.

 

  1. At the end of work:
  • Wash the vacuum rubber hose with 0.6% Sodium Hypochlorite.
  • Decontaminate solid and liquid waste as instructed below.
  • Wipe the hood, incubator handle and other equipment used (microscope, centrifuge, etc) with 70% Ethanol.
  • After removing the gloves, wash hands with soap and water (for at least 20 second).

Decontamination and spill procedures

Decontamination:

In general, decontamination is done using Sodium Hypochlorite. WIS warehouse offers a 6% Sodium Hypochlorite solution item number 020015987.

  • Keep in the hood a bottle of freshly prepared 0.6 % Sodium Hypochlorite (it is good for one week only)
  • Prepare 70% Ethanol sprayer

Solid waste decontamination:

All disposables (pipets, tips, flasks, plates) should be decontaminated in 0.6% sodium hypochlorite. After 30 min, discard the liquid in the sink and all de-contaminated solid waste in the biohazard bag. Biohazard bags used in the hood must be sealed (well but not too tightly) and transferred to the biohazard bag placed in the biohazard bucket. Dispose the bag into the departmental biohazard bin. Biohazard bags should be transferred inside the biohazard bin for disposal into the departmental biohazard bin.

Liquid waste decontamination:

Small volume liquid waste, up to 500 ml may be decontaminated in the biological hood using 500 ml plastic bottles (for example, medium or PBS bottles) containing 1:10 of the liquid waste volume of 6% Sodium Hypochlorite. Wait 30min and pour the decontaminated liquid to the sink. Pay attention not to dispose solids (like tips) to the sink, it is recommended to use a strainer.

Large volumes of liquid waste may be decontaminated using a vacuum pump/line protected by double trap system and 0.2 filter (Midisart® 2000 catalogue # 17805UPN, to be collected at the safety unit). The collection bottle should contain1:10 of the volume 6% Sodium Hypochlorite. Wait 30min and pour the decontaminated liquid to the sink.

Decontaminating a small volume spill -Cover the spill with paper towel and gently pour on top 0.6% Sodium Hypochlorite, starting from the perimeter inwards. Collect the paper towel to a biohazard bag.

Decontaminating a large volume spill: Wear a P3 respirator, a face protection shield and shoe covers, cover the spill with paper towel and gently pour on top 6% Sodium Hypochlorite (pour1/10 of the spill volume), starting from the perimeter inwards. Collect the paper towel to the biohazard bag. If needed dial 2999 for help.

In case of skin contact, wash twice the affected area with soap and water for at least 30 sec.  Call 2999 to report and, report accident also to the PI.

For eye exposure, flush with water for at least 15 minutes. Call 2999 to report, also report accident to the PI, seek medical advice.