FIG. 3.

Creation and characterization of Rep5 helper constructs for Cap1 to Cap5. (A) The AAV5 Rep and AAV1 to AAV4 Cap genes were subcloned into pXR2, a non-TR-containing plasmid (see Materials and Methods for details). These new helper plasmids were used to package TR5 vectors into Cap1 to Cap5. (B) Southern blot using a GFP-specific probe of Hirt DNA extracted from HEK 293 cells transfected with pRep5Cap1-5, an Ad helper plasmid, and TR5-GFP or TR2-GFP. The two replicative forms of the vector are indicated. (C) Cell lysate from triple transfections described above were used to infect naïve HEK 293 cells. Cells infected with lysate from TR5-eGFP vector transfection of capsid serotypes 1 to 5 were positive for GFP (panels 1 to 5 corresponding to AAV1 to AAV5). HEK 293 cells infected with lysate from TR2-eGFP vector transfection of capsid serotypes 1 to 5 did not express GFP (panel 6, AAV1; representative of AAV2 to AAV5). (D) Graph comparing the titers achieved in the production of TR2 versus TR5 rAAV. Titers of samples were determined in duplicate by quantitative PCR. Standard errors are indicated. (E) Graph comparing the transducing units per vector genome of rAAV TR2 versus TR5 vectors. Note that values on the y axis are multiplied by 1 × 10⁻⁷. Virus was serially diluted and used to infect cells. GFP-positive cells were quantitated and transducing units per microliter calculated before conversion to transducing units per vector genome. Samples were measured in duplicate, and standard errors are indicated.