FIG 5.

Broad interchangeability of AAP variants from multiple AAV serotypes. (A) Western blot analysis to confirm proper AAP expression from all constructs. HEK293T cells were transfected with the expression plasmids encoding AAP from AAV serotypes 1 to rh10 under control of a strong CMV promoter. AAP proteins were detected using an anti-HA antibody binding to an N-terminal HA tag present in all AAP expression constructs. The three lanes “mut” illustrate successful knockout of AAP following mutation of the start codon and introduction of a stop codon. To validate the effect of these changes, the mutated AAP sequences of AAV2, -8, and -9 (used as examples) were cloned into the identical expression plasmid as for all the wild-type AAPs. Note the complete absence of signals in these three lanes, verifying the full knockout. (B) trans-complementation assays in which AAP-deficient AAV1, AAV3 to -9, and AAVrh10 helpers were supplemented with their corresponding AAPs during vector production. The crude cell lysates produced were used for transduction of SF539 (AAV1, AAV3, AAV5 to -9, and AAVrh10) or MCF7 (AAV4) cells. Values were always normalized to the cognate wild-type (wt) controls (set to 1.0). (C) Cross-reactivities of 10 wild-type AAPs with the AAV2mut helper. The plasmid combinations shown were cotransfected to produce GFP-expressing vectors, which were then used to transduce HEK293T cells. Values were normalized to that for the wild-type AAV2 helper (set to 1.0). Shown are means from three independent biological replicates plus SD. Statistical analysis was by a one-way ANOVA with Bonferroni's multiple-comparison test (A/B). n.s., not significant; **, P < 0.01; ***, P < 0.001. (D) Sequence identity of AAPs of 10 different AAV serotypes. The heat map depicts the percentage of unique amino acids between a pair of two AAPs (see Materials and Methods for details). (E) trans-complementation of 10 AAP-depleted helper plasmids with 10 AAP variants. Shown are representative data from three independent crude lysate productions. In each row, the wild-type (wt) helper is set to 100%, and rescue potencies are color coded. Note that in some cases (‡), trans-complementation of the AAV3 mutant reached up to 150% of that of the wt helper.