Large commercial scale lentiviral vector production system and vectors produced thereby

A method for increasing the production of recombinant lentivirus (rLV) comprising a therapeutically beneficial transgene, comprising

• a) transfecting cells with plasmids encoding the necessary components for rLV production using a calcium chloride transfection mix, said calcium chloride and said plasmids forming a complex, said complex being added to said cells at a uniform constant speed;
• b) incubating said cells for 56 hours, wherein virus particle containing media is collected at 32 hours, replaced with fresh media which is collected at 56 hours, said collected media being placed in a cold storage unit, thereby reducing virus inactivation;
• c) concentrating the viral particles in said media via tangential flow filtration;
• d) treating said particles with an enzyme which reduces nucleic acid contamination;
• e) centrifuging the treated particles of step d) to further concentrate said rLV;
• and
• f) layering the concentrated rLV of step e) over a sucrose gradient, thereby producing rLV at high yield.