Generation and characterization of chimeric recombinant AAV vectors

Abstract

Although most animal experiments with recombinant adeno-associated virus (AAV) vectors have been based on AAV serotype 2, recent studies showed that AAV vectors based on AAV serotype 1 performed more efficiently in muscle and other tissues. On the other hand, AAV2-based vectors can be readily purified by heparin column. To combine the advantages of both types of vectors, we developed a strategy to generate chimeric vectors by using a mixture of AAV helper plasmids encoding both serotypes in the transfection process. Because the AAV packaging machinery cannot distinguish between closely related AAV1 and AAV2 capsid proteins, each packaged virion contains capsid proteins from both serotypes. As expected, the resulting chimeric vectors could be purified by heparin column. Neutralizing antibody assays showed that the chimeric vectors can be inhibited by either AAV1 or AAV2 antiserum. In vivo, the chimeric vectors direct levels of expression similar to those of AAV1 in muscle or AAV2 in liver; that is, they combine the best transduction characteristics of both parent vectors. In summary, this study provides a straightforward method for combining various properties of different AAV serotypes into one vector. Potential limitations of the chimeric vectors are also discussed.

FIG. 1

(A) An illustration of the strategy to produce chimeric recombinant AAV vectors based on AAV1 and AAV2. AAV1 and AAV2 genomes and expressed proteins are shown in different colors. The mRNAs for Rep78, 68, 52, and 40 and VP1, 2, and 3 are indicated. Promoters P5, P19, and P40 are marked by arrows. (B) The helper effects on the composition of chimeric AAV vectors. The hypothetical composition of vector capsid is calculated based on the different ratios of helper plasmids used for transfection. A total of 50 μg DNA was used to transfect a 15-cm dish of 293 cells. For example, AAV12_1:9 can be made by transfecting 5 μg AAV1 helper and 45 μg AAV2 helper along with adenovirus miniplasmid and vector plasmid into 293 cells.

FIG. 2

Production of chimeric vectors. AAV1 and AAV2 chimeric vectors were generated by cotransfection of pAAV.F.IX, pAd, and AAV helper into 3 × 10⁸ 293 cells. The ratios of AAV1 and AAV2 helper are 10:0, 9:1, 1:1, 1:9, and 0:10. The vectors were purified by CsCl gradient. Vectors at the density of 1.38 -1.42 g/ml were collected. The titer was determined by quantitative PCR. The experiment was repeated three times and similar results were obtained. The y axis shows the total amount of vectors isolated in genomes. The standard deviation is identified.

FIG. 3

Purification of chimeric vectors by heparin column. The AAV1 and AAV2 chimeric vectors were generated by cotransfection of pAAV.F.IX, pAd, and AAV helper into 3 × 10⁸ 293 cells. The ratios of AAV1 and AAV2 helper are 10:0, 9:1, 1:1, 1:9, and 0:10. The vectors were purified by using 1.5 g heparin resin per sample. Vectors were eluted by a NaCl gradient ranging from 200 mM to 1 M. The elution volume for each fraction was 10 ml. The vector titer was determined by quantitative PCR. The experiment was repeated three times. Shown is the vector distribution in the elution gradient. The y axis shows vector titer (genomes/ml) in each fraction. To obtain the total vector yield, it must be multiplied by a factor of 10. The x axis is the salt concentration used for elution. The mean yield and standard deviation in each fraction are calculated from three independent experiments.