1. ¹Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA
2. ²Department of Medicine, University of Hong Kong, Hong Kong, Hong Kong
3. ³Shanghai Institute of Hematology, Ruijin Hospital Affiliated to Shanghai Second Medical University, Shanghai, China

Abstract

Adeno-associated virus (AAV) serotype 1 (AAV1) has been shown to be more effective than the well studied AAV serotype 2 (AAV2) in muscle. In our previous study to identify muscle specific determinants on the AAV1 capsid, a series of AAV capsid mutants were generated to identify the major regions affecting AAV transduction efficiency in muscle (J Virol. 77:2768-74, 2003). Replacement of amino acids 350 to 430 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector that behaved similarly to AAV1 in vitro and in vivo in muscle. Intramuscular injection of 1|[times]|10e11 vector particles/mouse of hybrid vector carrying a human FIX transgene in CD4 KO mice resulted in levels of human FIX in the plasma of around 450 ng/ml, which were more than 4-10 fold higher than mice injected with an AAV2 vector carrying the same transgene, and reached about 75% transgene levels of AAV1 injected animals. DNA analysis of injected muscle showed 10 fold higher copy number after gene delivery with the hybrid vector compared to 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 not only have an effect on virus-cell receptor interaction, but also influence trafficking and processing of the virus particle in the cell.

This |[ldquo]|Hybrid Vector|[rdquo]| retains the Heparin binding sites of AAV2 and therefore can be purified by a heparin-sepharose column with the same efficiency as AAV2. When tested in vivo, either in CD4 KO mice or in a hemophilic mouse model, the heparin purified hybrid vector showed a more than 10 fold higher activity than heparin-sepharose purified AAV2. This demonstrates the utility of this hybrid vector in large scale heparin-column purification generating a vector with a high expression profile for muscle directed gene delivery. Since it is only 1% different from the vector already evaluated in clinical trials of AAV2 vectors, it may be readily introduced into clinical studies.