Efficient AAV1-AAV2 hybrid vector for gene therapy of hemophilia.
Adeno-associated virus (AAV) serotype 1 (AAV1) has been shown to be more effective than the well-studied AAV serotype 2 (AAV2) in muscle gene transfer. Replacement of amino acids 350 to 430 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector, termed AAV-221-IV, which behaved similarly to AAV1 in vitro and in vivo in muscle. Intramuscular injection of 1x10(11) vector particles per mouse of hybrid vector carrying a human FIX transgene in CD4 knockout mice resulted in an average level of human FIX in the plasma of 450 ng/ml, 4- to 10-fold higher than in mice injected with an AAV2 vector carrying the same transgene, and 80% of the transgene levels in animals treated with the same dose of AAV1. DNA analysis of injected muscle showed a 10-fold higher copy number after gene delivery by the hybrid vector compared with AAV2. A comparison of total DNA versus DNA from intact virus particles suggests a higher stability of hybrid virus particles. These results suggest that changes in the AAV capsid have an effect on virus-cell receptor interaction, and also influence trafficking and processing of the virus particle in the cell. This "hybrid vector" retains the heparin-binding sites of AAV2 and, therefore, can be purified by passage through a heparin-Sepharose column with the same efficiency as AAV2. When tested in vivo, either in CD4 knockout mice or in a hemophilic mouse model, the heparin-purified hybrid vector showed >10-fold higher activity than similarly purified AAV2. This demonstrates the utility of this hybrid vector in the performance of large-scale heparin column purification to generate a vector with a high expression profile for muscle-directed gene delivery. Initiation of clinical studies with this hybrid vector may be facilitated because it differs from AAV2 by only nine amino acids.
Hauck
Bernd
B
Department of Pediatrics, University of Pennsylvania Medical Center and Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Xu
Ray Ruian
RR
Xie
Jing
J
Wu
Wenman
W
Ding
Qiulan
Q
Sipler
Matthew
M
Wang
Hongli
H
Chen
Ling
L
Wright
J Fraser
JF
Xiao
Weidong
W
D000818:Animals; D019556:COS Cells; D036022:Capsid Proteins / Q000235:genetics; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000229:Dependovirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D005164:Factor IX / Q000032:analysis / Q000096:biosynthesis; D015870:Gene Expression / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000592:standards*; D006367:HeLa Cells; D002836:Hemophilia B / Q000628:therapy*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D010641:Phenotype; D017386:Sequence Homology, Amino Acid; D014161:Transduction, Genetic
D036022:Capsid Proteins; D005164:Factor IX