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

Abstract

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 smaller than 4.1 kb, the packaging efficiency was also suboptimal. In contrast, when the size of the genome was less than half the length of the wild-type genome, two copies of the vector were packaged into each virion, suggesting that the copy number control during packaging is a “head-full” mechanism. Because the length of the minimal cDNA of CFTR is about 4.5 kb, these results suggest it is possible to package the CFTR gene into AAV if the combined length of transcriptional elements and ITRs is kept under 500 bp. The results of this study are important for directing the design of AAV vectors for efficient gene transfer, as well as for a better understanding of the mechanism of AAV genome packaging. Adeno-associated virus (AAV) has been engineered as vectors to express genes of interest. The genome size of wild-type AAV is 4.6 kb. The total of size of the AAV vector influences the efficiency of its packaging into AAV virions. We have quantitatively determined the relationship of vector size to packaging efficiency by constructing 12 AAV vectors of different lengths. These vectors vary from 2 kb to 6 kb in size and carry a chloramphenicol acetyltransferase (CAT) reporter gene. The packaging efficiencies of these vectors were determined both by quantitating the DNA content of viral particles and assaying the efficiency of AAV virions in transferring CAT gene into HeLa cells. The results of our study demonstrate that the packaging efficiency of AAV is greatly affected by the length of the genome. AAV can package vectors ranging from 2 to 5.2 kb in size, but the optimal vector length for packaging is between 4.1 and 4.9 kb.