Figure 3.

Validation of Selected rAAV vectors in transwell, hiPSC-derived cortical neurons and astroglia cells, and mice

(A) Fourteen AAV capsids from the three selections were vectorized and tested in the transwell BBB system with hCMEC/D3 and primary astrocytes, each rAAV was tested in separate transwells and viral genome copy numbers as a percentage of input virus were determined in flowthrough media at 3, 6, and 24 h and in astrocytes at 24 h. (B) Transduction efficiency of 12 AAV capsids from the three selections packaged with firefly luciferase expression cassette were tested in hiPSC-derived cortical neurons and astroglia cells. Relative luciferase activity as compared with AAV9 in each cell type were determined after 72 h. (C) Delivery of vector genomes and expression of luciferase in mouse liver and brain after retro-orbital injection was determined for RS.N8d, RS.R5, RS.A5e, and RS.A6 at 21 days and for RS.R3, RS.R6, RS.R11, and RS.R18 at 28 days. n ≥ 3 for all conditions, significance determined by the Student t test; ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.