Figure 3.

AAVR Is Essential for Entry of GluA4-AAV

(A) Model of a GluA4-AAV^R513A vector particle is shown. DARPins (red) are extruding from pores at the 5-fold symmetry axis of the AAV capsid (left). Point mutations inserted in the capsid are shown in red and orange on the blow-up of the 3-fold axis (right). The illustration was created using Pymol. Adapted from Münch and colleagues.¹⁹ (B) GluA4-targeted vectors harboring two (R585A and R588A; GluA4-AAV) or three point mutations (R585A, R588A, and R513A; GluA4-AAV^R513A) transduce GluA4-positive CHO-GluA4 cells. Cells were incubated with the indicated AAV vectors encoding GFP at a GOI of 1.25 × 10⁶ (GluA4-AAV, GluA4-AAV^R531A) or 1 × 10⁴ (AAV2). Cells were analyzed 72 h after transduction by flow cytometry. Untransduced cells were used as control. (C) Reduced off-target transduction with GluA4-AAV^R513A. GluA4-positive or -negative cells of the indicated cell lines were incubated with GluA4-AAV_mut or GluA4-AAV_mut^R513A vectors encoding GFP at a GOI of 7.9 × 10⁵ (CHO) or 2.6 × 10⁵ (SH-SY5Y and HT1080). Cells were analyzed 72 h after transduction by flow cytometry. Each transduction experiment was performed in technical triplicates. The mean and SD are shown. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05. ns, not significant by unpaired t test. Representative dot plots are shown in Figure S3. (D) AAV vectors transduce AAVR-positive cell lines only. AAVR-positive and -negative cell lines were incubated with the indicated vector stocks encoding GFP at a GOI of 4.9 × 10⁵ (GluA4-AAV, GluA4-AAV^R531A, Her2-AAV) or 1 × 10⁴ (AAV2). Cells were analyzed 72 h after transduction by flow cytometry. Each transduction experiment was performed in technical triplicates. Mean value and SD are shown. (E) GluA4-AAV vector particles bind to AAVR-positive and -negative GluA4-expressing cells. The indicated cell lines were incubated with GluA4-AAV^R513A at a GOI of 3 × 10⁴ for 1 h at 4°C. Bound particles were stained with an AAV capsid-specific antibody in combination with a PE-conjugated secondary antibody. Mean PE fluorescence intensity (MFI) was measured via flow cytometry. An unrelated vector targeted to Her2 (Her2-AAV; GOI 8 × 10⁴) was used as control. Each binding experiment was performed in technical triplicates. Mean value and SD are shown. ***p ≤ 0.001. ns, not significant by unpaired t test.