Lentiviral vector production is complex. Circular DNA plasmids are produced which encode the structural components of the virus required to package the viral single stranded RNA into the final vector. The entire production process takes place inside human cells grown in culture.
Transfection of Expression Plasmids
The plasmids encoding the viral coat proteins, the viral central core components and the lentiviral vector RNA genome are transfected into human cell culture. They are imported into the cell nucleus and start expressing the genes they encode.
The envelope proteins are ultimately found on the surface of the lentiviral vector particles. The envelope proteins determine the lentiviral vectors tissue specificity. In our case we use modified versions of the Sendai Virus F & HN proteins to target our vector to lung cells. The envelope proteins congregate at the cell surface of the mammalian cells producing the vector.
Changing a Virus Pseudotype
Basic description of Lentivirus Pseudotyping
The viral central core proteins and the lentiviral vector RNA are ultimately found inside the lentiviral vector particles. These crucial components allow the vector to introduce its therapeutic gene into the targeted lung cells. During vector production, several viral proteins and two copies of the lentiviral vector RNA genome come together inside the producer cell to produce inactive, intermediate forms of the vector known as virions.
Budding of the Virion
The viral virions fuse with the cell surface membrane of the production cell and bud off into the mammalian cell culture medium. This process ensures that the final, active lentiviral vector particles are surrounded by a lipid bilayer which contains the envelope proteins.
Various filtration and purification methods based on the size (they are smaller than mammalian cells, but larger than all the cell components) and negative charge of the lentiviral vector particles are used to process the cell culture media. This produces a final lentiviral vector particles free of contaminants, other cell products or the plasmid molecules.