Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.
Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.
Hermens
W T
WT
Graduate School for Neurosciences Amsterdam, Netherlands Institute for Brain Research.
ter Brake
O
O
Dijkhuizen
P A
PA
Sonnemans
M A
MA
Grimm
D
D
Kleinschmidt
J A
JA
Verhaagen
J
J
D000818:Animals; D001921:Brain; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002586:Cesium / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry; D003287:Contrast Media / Q000737:chemistry; D000229:Dependovirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000302:isolation & purification*; D007150:Immunohistochemistry; D009420:Nervous System; D051381:Rats; D013116:Spinal Cord; D014283:Triiodobenzoic Acids / Q000737:chemistry
D002712:Chlorides; D003287:Contrast Media; D014283:Triiodobenzoic Acids; D002586:Cesium; C028019:cesium chloride; C044834:iodixanol