Packaging of an AAV vector encoding human acid alpha-glucosidase for gene therapy in glycogen storage disease type II with a modified hybrid adenovirus-AAV vector.
We have developed an improved method for packaging adeno-associated virus (AAV) vectors with a replication-defective adenovirus-AAV (Ad-AAV) hybrid virus. The AAV vector encoding human acid alpha-glucosidase (hGAA) was cloned into an E1, polymerase/preterminal protein-deleted adenovirus, such that it is packaged as an Ad vector. Importantly, the Ad-AAV hybrid cannot replicate during AAV vector packaging in 293 cells, because of deletion of polymerase/preterminal protein. The residual Ad-AAV in the AAV vector stock was reduced to <1 infectious particle per 10(10) AAV vector particles. These modifications resulted in approximately 30-fold increased packaging of the AAV vector for the hybrid Ad-AAV vector method as compared with standard transfection-only methods. Similarly improved packaging was demonstrated for pseudotyping the AAV vector as AAV6, and for AAV vector packaging with a second Ad-AAV vector encoding canine glucose-6-phosphatase. Liver-targeted delivery of either the Ad-AAV hybrid or AAV vector particles in acid alpha-glucosidase-knockout (GAA-KO) mice revealed secretion of hGAA with the Ad-AAV vector, and sustained secretion of hGAA with an AAV vector in hGAA-tolerant GAA-KO mice. Further development of hybrid Ad-AAV vectors could offer distinct advantages for gene therapy in glycogen storage diseases.
Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Baodong
B
Sun
Y-T
YT
Chen
Andrew
A
Bird
Fang
F
Xu
Yang-Xun
YX
Hou
Andrea
A
Amalfitano
Dwight D
DD
Koeberl
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D003673:Defective Viruses / Q000235:genetics; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D004285:Dogs; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage; D006009:Glycogen Storage Disease Type II / Q000378:metabolism / Q000628:therapy*; D006367:HeLa Cells; D006801:Humans; D007275:Injections, Intravenous; D051379:Mice; D018345:Mice, Knockout; D011169:Portal Vein / Q000378:metabolism; D019065:Virus Assembly; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism
D000520:alpha-Glucosidases