Figure 1.
Analysis of EGFP expression after transduction of HEK293 cells with individual site-directed AAV2 capsid mutants.
Each of the 17 surface-exposed threonine (T) residues in AAV2 capsid was substituted with valine (V) and evaluated for its efficiency to mediate transgene expression. (a) EGFP expression analysis at 48 h post-infection at MOI of 1×103 vg/cell. (b) Quantification of transduction efficiency of each of the threonine-mutant scAAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2.
Figure 2.
Analysis of EGFP expression in HEK293 cells infected with multiple site-directed AAV2 capsid mutants.
Several most efficient threonine mutations were combined on single AAV2 capsid to produce double- and triple-mutant and efficiency of each vector was evaluated. (a) EGFP expression analysis at 48 h post-infection at MOI of 1×103 vg/cell. (b) Quantification of transduction efficiency of each of the threonine-mutant AAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2.
Figure 3.
Evaluation of EGFP expression in H2.35 cell transduced with capsid optimized AAV2 vectors.
The most efficient tyrosine, serine and threonine mutations were combined on single AAV2 capsid to produce several optimized AAV mutants. Efficiency of each vector was estimated on immortalized murine hepatocytes. (a) EGFP expression analysis at 48 h post-infection at MOI of 1×103 vg/cell. (b) Quantification of transduction efficiency of each of the optimized scAAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2.
Figure 4.
Kinetics of EGFP expression in H2.35 cell mediated by capsid optimized AAV vectors.
(a) EGFP expression analysis at 16, 24 and 48 h post-infection at MOI of 1×103 vgs/cell. (b) Quantification of transduction efficiency of each of the optimized scAAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2.
Figure 5.
Analysis of intracellular trafficking of AAV multiple mutant vectors to the nucleus.
Nuclear and cytoplasmic fraction of H2.35 cell infected with AAV2-WT, AAV2-Y444+500+730F and AAV2- Y444+500+730F+T491V mutant were separated and qPCR analysis was performed to evaluate vector genome distribution within cell in 16 h (a) and 48 h (b) post infection. **P<0.001 vs. WT in nucleus was considered as significant.
Figure 6.
In vivo imaging of luciferase gene expression following tail vein injection of multiple site-directed AAV2 capsid mutants.
C57BL/6 mice were injected with 1×10e10 vg/animal of several most efficient mutant scAAV vectors carrying luciferase gene. Live images were taken to analyses difference in luciferase activity. The visual output represents the number of photons emitted/second/cm2 as a false color image where the maximum is red and the minimum is blue (a) and relative signal intensity (b). *P<0.005 was considered as significant.
Figure 7.
(a) The capsid surface of AAV2 (grey) with the 17 surface threonine residues mutated in blue (251, 329, 330, 454, 503, 581, 592, 597, 660, 671, 701, 713, 716), green (455), yellow (491), brown (550), and pink (659). The surface location of T329, T330, T713 and T716 are indicated by arrows. The five-fold symmetry related DE loops (between the βD and βE strands) are colored in orange. The HI loops (between the βH and βI strands) are colored white and S662 located in this loop is in red. The white dashed triangle in (a) depicts a viral asymmetric unit bounded by a five-fold axis and two three-fold axes with a two-fold axis between the three-folds. Dashed ovals delineate the approximate footprints (2/60) of threonine residues that affect transduction when mutated. (b) A “Roadmap” projection [23] of the AAV2 capsid surface residues within a viral asymmetric unit. The areas covered by AAV2 surface threonines and S662 are colored as in (a). The residues in the tyrosine triple mutant residues, 444, 500, and 730 are shown in shades of purple. Dashed ovals are as described in (a). Dashed rectangle (blue) shows residues previously determined to be important in heparin sulfate receptor binding for AAV2 and AAV6 [37], [50].