Figure 3.
In Vitro Functional Characterization of Provectors
(A) Proteolytic cleavage fragments of provector capsid subunits can be detected via silver stain. The vectors were treated with MMP-2, -7, -9, or sham buffer (S). Intact VPs are observed in the sham conditions. Conversely, N-terminal (VP1′, VP2′, and VP3′) and C-terminal (C-term) fragments are observed for L001 following treatment with MMPs. AAV9 and scrambled vectors do not yield VP cleavage fragments in response to MMPs. (B) Provectors demonstrate protease-activated transduction behavior. Vectors were treated with sham buffer or MMPs and then added to CHO-Lec2 cells (MOI: 5,000). Flow cytometry was then used to quantify GFP expression at 48 h post-transduction. The transduction index (TI) is calculated as the percent of GFP-positive cells multiplied by the geometric mean fluorescence intensity of the GFP-positive cells (TI = %GFP × gMFI). TI is a linear indicator of virus activity over a wider range of MOIs compared to the percent of GFP-positive cells alone.12 AAV9 demonstrates high transduction efficiency and is not affected by MMPs. L001 and L005 both demonstrate significantly higher transduction (∼5× and ∼2×, respectively) when treated with MMPs compared to sham treatment. Scrambled vector demonstrates low transduction efficiency regardless of MMP treatment. Error bars, SEM; n = 3; *p < 0.05, **p < 0.001. (C) Lectin competition assay demonstrates switchable galactose-binding activity by provector. Vectors were treated with either MMP-9 or sham buffer, then mixed with Erythrina cristagalli lectin and incubated with CHO-Lec2 cells (MOI: 5,000) for 1 h at 4°C. Media were removed, cells were washed with PBS, and fresh media were added. 48 h later, transduction was quantified using flow cytometry. AAV9 transduction is blocked by a high concentration of lectin, regardless of MMP treatment. In the locked (sham) state, L001 has a low transduction efficiency. After MMP exposure, L001 transduction is restored and is competitively inhibited by lectin. Results from two independent experiments are plotted for each lectin concentration, with the horizontal line indicating the average.