Relationship between retroviral vector membrane and vector stability.
The present work studies the physico-chemical properties of retroviral vector membrane, in order to provide some explanation for the inactivation kinetics of these vectors and to devise new ways of improving transduction efficiency. For this purpose, vectors with an amphotropic envelope produced by TE Fly A7 cells at two culture temperatures (37 and 32 degrees C) were characterized by different techniques. Electron paramagnetic resonance (EPR) results showed that vectors produced at 32 degrees C are more rigid than those produced at 37 degrees C. Further characterization of vector membrane composition allowed us to conclude that the vector inactivation rate increases with elevated cholesterol to phospholipid ratio. Differential scanning calorimetry (DSC) showed that production temperature also affects the conformation of the membrane proteins. Transduction studies using HCT116 cells and tri-dimensional organ cultures of mouse skin showed that vectors produced at 37 degrees C have higher stability and thus higher transduction efficiency in gene therapy relevant cells as compared with vectors produced at 32 degrees C. Overall, vectors produced at 37 degrees C show an increased stability at temperatures below 4 degrees C. Since vector membrane physico-chemical properties are affected in response to changes in culture temperature, such changes, along with alterations in medium composition, can be used prospectively to improve the stability and the transduction efficiency of retroviral vectors for therapeutic purposes.
Carmo
M
M
IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal.
Faria
T Q
TQ
IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal.
Falk
H
H
Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Coroadinha
A S
AS
IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal.
Teixeira
M
M
IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal.
Merten
O-W
OW
Généthon, 1 bis Rue de l'Internationale, F-91000 Evry, France.
Gény-Fiamma
C
C
Généthon, 1 bis Rue de l'Internationale, F-91000 Evry, France.
Alves
P M
PM
IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal.
Danos
O
O
Généthon, 1 bis Rue de l'Internationale, F-91000 Evry, France.
Panet
A
A
Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Carrondo
M J T
MJT
Laboratório de Engenharia Bioquímica, FCT/UNL, P-2825 Monte da Caparica, Portugal.|IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal.
Cruz
P E
PE
Universidade Atlântica, Antiga Fábrica da Pólvora de Barcarena, P-2745-615 Barcarena, Portugal.|ECBio, Lab. 4.11, Ed. ITQB, Apartado 98, P-2781-901 Oeiras, Portugal.|IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal.
D000818:Animals; D002151:Calorimetry; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism* / Q000821:virology; D004578:Electron Spin Resonance Spectroscopy; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D011487:Protein Conformation; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014161:Transduction, Genetic; D038301:Virus Inactivation
D008565:Membrane Proteins