Quantitative Analysis of the Packaging Capacity of Recombinant Adeno-Associated Virus

作者：Jian-Yun Dong, Peidong Fan, Raymond A. Frizzell · 发表于：Human Gene Therapy · 年份：1996 · DOI：10.1089/hum.1996.7.17-2101 · 被引用次数：592 · 研究领域：Virus-based gene therapy research、Cystic Fibrosis Research Advances、CRISPR and Genetic Engineering

Recombinant adeno-associated viruses (AAV) are among the most promising vectors for gene therapy of genetic diseases, including cystic fibrosis (CF). However, because of its small genome size, the capacity of AAV to package a therapeutic gene is limited. The efficiency of packaging the cystic fibrosis transmembrane conductance Regulator (CFTR) gene into AAV will be an important factor in determining whether recombinant AAV can be developed as a vector for transferring CFTR cDNA to the airway epithelia of patients with CF. Current understanding of the AAV biology suggests that AAV can package a genome slightly larger than the size of a wild-type genome. The precise range of the genome size and the efficiency of packaging have not been defined. Using a series of AAV vectors with progressively increasing genome size, we were able to analyze quantitatively the packaging efficiency in relation to the vector size and to determine the size limit for packaging. The packaging efficiencies of AAV vectors of variable sizes were determined directly by assaying DNA contents of viral particles, and indirectly by analyzing their efficiency in transfer of a chloramphenicol acetyltransferase (CAT) reporter gene into target cells. Our studies showed that the optimal size of AAV vector is between 4.1 and 4.9 kb. Although AAV can package a vector larger than its genome size, up to 5.2 kb, the packaging efficiencies in this large size range were sharply reduced. When the AAV genome size was small...