FIGURE 4.

Transduction, binding, and uptake of the AAV9NGA mutant in CHO Lec2 cells. A, transduction efficiency of AAV9-CBA-Luc (white columns) and AAV9NGA-CBA-Luc (black columns) on CHO Lec2 cells that contain surface-exposed galactose essential for AAV9 infection at increasing multiplicities of infection were determined 24 h post infection (n = 4). B, sialic acid-deficient CHO Lec2 cells were prechilled and incubated with AAV9 or AAV9NGA particles at different multiplicities of infection ranging from 100–500,000 at 4 °C to allow binding without cellular uptake. Quantitative analysis of dose-dependent AAV9 (solid circles) or AAV9NGA (triangles) attachment to cell surfaces was assessed using qPCR to quantify viral genome copy number and binding curves generated using a single-site binding model (n = 5). The inset shows data of capsid binding at earlier time intervals. C, for cellular uptake studies following removal of unbound virions, viral genomes were extracted from Lec2 cells either 5 (gray columns) or 60 min (white columns) after incubation, and copy numbers were determined by qPCR. The percentage of internalized virions is derived from the ratio of total number of internalized virions normalized to the number of bound AAV9, AAV9NGA, or the binding deficient AAV9/W503R virions per cell (n = 5). Error bars represent mean ± S.E. *, p < 0.05; ***, p < 0.005. RLU, relative light unit(s).