We performed characterization of recombinant vectors of serotype 9 adeno associated virus (rAAV) with methods such as digital droplet polymerase chain reaction (ddPCR), quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM). We studied four different fractions (heavy, full, intermediate, empty) of two types of vectors, single-stranded AAV (ssAAV) and self-complementary AAV (scAAV). Fractions were collected after purification by density gradient ultracentrifugation with CsCl. We expected that characterization of viral vectors with different methods would result in determination of the differences between individual fractions. We expected the highest concentration of full capsids and the lowest content of impurities in full fraction. We expected that individual methods would correlate with each other according to the type of vector. Our study showed differences in the composition of individual factions, although they were not as high as expected. The lowest content of impurities was determined in full fraction. The highest content of impurities was determined in heavy and empty fractions. We showed that full fraction contains the highest concentration of full capsids if ssAAV vectors are used. On the other hand, full and intermediate fractions contain the highest concentration of full capsids if scAAV vectors are used. The highest percentage of empty particles was determined in empty fraction for both types of vectors. There were smaller differences between fractions with scAAV, which contained higher values of capsid and viral genome titers and lower concentrations of impurities compared to ssAAV. We have shown that the methods complement each other and that there are correlations between them. In conclusion, the use of various approaches at different levels is crucial for characterization of viral vectors for gene therapy.
|
