Figure 1.

Programming AAV capsid with proteolytic regulation. (a) Operation of PAVs. Locked virus cannot interact with cellular HSPG until processed by target protease. Upon virus activation, genetic payload (CMV-GFP transgene) is delivered to cell nucleus. (b) Molecular models of PAV1 (Table 1) in locked (gray) and unlocked (blue) states. Peptide lock leaving group in red. (c) Electron micrographs of PAV1 in the locked (left) and unlocked (right) states indicate capsids remain intact after MMP-7 treatment (scalebar = 20 nm). (d) Western blot with B1 antibody shows cleaved VP after MMP-7 (+) treatment compared to sham (−). (e) Heparin chromatography reveals low-affinity uncleaved virus (gray) elutes in low salt, whereas MMP-7 treated particles require high ionic strength to disrupt tight AAV-heparin interactions. (f) HEK293T cells transduced (gMOI = 700) by PAV1 capsids, packaging a single-stranded (ssDNA) or a self-complementary double-stranded (dsDNA) transgene,³⁰ show striking increases in gene delivery after treatment by MMP-7 (blue bars) compared to sham (gray bars). Expectedly, the dsDNA transgene increases gene expression from both sham and MMP-7 treated PAV1. Wt capsids shown for comparison (note: dsDNA is saturated at approximately 100% GFP+ cells). (g, h) MMP-7 treated PAV at right, sham at left. (g) Fluorescent micrographs of HEK293T cells transduced with PAV1 (ssDNA) before/after MMP-7 treatment. (h) Dotplots of HEK293T cells transduced by PAV12 (dsDNA, gMOI = 1000). Error bars are SEM from two (panel e) and three (panel f) independent experiments, each performed in duplicate.