Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography.
BACKGROUND
During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly.


METHODS
Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography.


RESULTS
Our results show that unconcentrated LV vector stocks with titers in excess of 108 transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm2 tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 x 1010 TU were recovered from a single HYPERFlask vessel.


CONCLUSIONS
The protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols.
Kutner
Robert H
RH
Gene Therapy Program, Vector Core, Louisiana State University Health Sciences Center, New Orleans, LA, USA. rkutne@lsuhsc.edu
Puthli
Sharon
S
Marino
Michael P
MP
Reiser
Jakob
J
D000837:Anion Exchange Resins; D018929:Cell Culture Techniques / Q000379:methods; D002845:Chromatography / Q000379:methods*; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000302:isolation & purification*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006367:HeLa Cells; D006801:Humans
D000837:Anion Exchange Resins