Lentiviruses

Lentiviruses, a genus within the Orthoretroviridae subfamily of the Retroviridae family, are known for their long incubation periods, hence the name Lentivirus, derived from “lente-”, the Latin word for “slow”.

Overview

These viruses are enveloped and spherical, with a diameter ranging from 80 to 100 nm. The envelope, which is a lipid membrane, originate from the host cell membrane, including phospholipids and proteins, but also contains viral glycoproteins. Inside this envelope is the nucleocapsid or core, which houses the single stranded viral RNA. A distinctive feature of lentiviruses, and indeed of the Retroviridae family, is their ability to infect both dividing and non-dividing cells. They can also integrate a substantial amount of viral RNA into the host cell’s DNA, making them one of the most effective methods for gene delivery.

  • Require BSL-2 containment.
  • Can theoretically carry payload up to 9 kb (10 and 11 kb have also been reported)
  • Safety concerns regarding insertional mutagenesis.
  • Use of vesicular stomatitis virus G glycoprotein (VSV-G) allows a broad host range, ultracentrifugation concentration, and high titers. However, it can have cytotoxic effects on producing cells, so viruses are obtained by transient transfection.
  • Production is achieved through trans-complementation, using 2nd and 3rd generation vectors.
    Lentiviruses transduce most cell types within the central nervous system (CNS) in vivo.
Lentiviral Vector Generations 2023.
Figure 1: Genome of HIV-1 and the evolution to third generation of lentiviral vector systems. Image: Marc Panas

Lentiviruses are created by introducing various plasmids into production cell lines, such as HEK293 Lenti-X® (as shown in the figure). This method enhances biosafety significantly by minimizing the risk of generating replication-competent lentiviruses. This is achieved by separating the cis-acting sequences (found in the transgene plasmid in the figure), which are necessary for transferring the viral genome to target cells, from the trans-acting sequences that encode the viral proteins (gag, pol, and env VSV-G). Both types of constructs are introduced into the same cell to produce the virions. We are producing viruses based in the 2nd generation packaging systems and 3rd generation packaging this means that all accessory genes have been deleted and the genes necessary for viral replication (pol), viral surface proteins (env: VSV-G) and viral core proteins (gag) are provided in separate constructs. One advantage of this system is that the packaging and envelop vectors work with 2nd and 3rd generation transgene vectors, offering a broader range of possibilities.

See detailed description of Figure 1.

Production and services

Packaging plasmids of 2nd and 3rd generation (provided by two or three different plasmids, which are capable of packaging both 2nd and 3rd generation transfer plasmids. All our lentiviruses are produced using the VSV-G envelop to concentrate the viruses by ultracentrifugation and obtain high titers adequate for in vivo experiments.

Services include:

  • Transfection and supernatant collection
  • Concentration by two-step centrifugation, to suitable volumes specified by the customer.
  • Quality control assay includes a physical titer (vRNA copies/mL) by qRT-PCR or by lateral flow device measuring lentiviral p24. Functional titer (IFU/mL) by transduction of cell lines and analysis by FACS when reporter genes (e.x., GFP) are present in the expression vector. Alternatively, titration based on proviral DNA copies present in genomic DNA extracted from transduced cells. The titration is requested by the customer.

Note: For a successful virus production, the VirusTech Core Facility recommends transforming the expression plasmids into E. coli strains recommended for use when cloning unstable inserts such as lentiviral DNA containing repeat elements. Such strains can be obtained from different companies such One Shot™ Stbl3™ #C737303 (Thermo Fisher Scientific) or NEB Stable competent E. coli #C3040I (NEB).

Price list

Higher titer production of up to ≥ 1*1010 IFU/ml (1*1012 vg/ml) is possible, please contact us for consultation.

For KI users
Product (KI users) Prices (1) in SEK Volume Titers in IFU/mL(vg/mL) Timeline (2)
Non-concentrated supernatant 4700 120 ml ≥ 1*105 - 1*106 3 weeks
Concentrated 5700 500-1000 µl ≥ 1*108 (1*1010) 4 weeks
Ultraconcentrated 7400 50-70 µl ≥ 1*109 (1*1011) 4 weeks
For external Users
Product (external Users) Prices (1) in SEK Volume Titers in IFU/mL(vg/mL) Timeline (2)
Non-concentrated supernatant 5500 120 ml ≥ 1*105 - 1*106 3 weeks
Concentrated 8000 500-1000 µl ≥ 1*108 (1*1010) 4 weeks
Ultraconcentrated 8900 50-70 µl ≥ 1*109 (1*1011) 4 weeks
Titration method
Titration method Prices (internal) in SEK (1) Prices (external) in SEK (2)
Transduction + FACS (functional) 3300 4100
Transduction + Provirus qPCR (functional) 3900 4700
qRT-PCR (physical) 3100 3600
Lenti-X GoStix (physical) 700 800

(1) Prices include INDI

(2) From the time the plasmid is received depending on the current queue.

Ordering Lentivirus production

  1. Download the Lentivirus order form
  2. Fill it out an submit to Marc Panas at marc.panas@ki.se
MP
Content reviewer:
31-01-2024