Consequences of human genetic variations in KIAA0319L, encoding adeno-associated virus receptor, on AAV-mediated gene transfer

Figure 1. Structure of AAVR and location of human AAVR nonsense and missense variants evaluated in this study The AAVR N and C termini are cytoplasmic, creating an extracellular peptide loop. The extracellular portion includes five PKD domains including PKD1 and PKD2 implicated in AAV interaction for most AAV serotypes.20,21,26,27,28,29 The cytoplasmic C terminus of AAVR contains endocytic trafficking consensus peptide sequences including a YXXΦ motif, a di-leucine motif, and an acidic cluster, all of which have a role in clathrin-mediated endocytosis.58,59 Red octagons represent nonsense variants. Yellow circles are missense variants. See Table 1 for details relating to the AAVR variants assessed.

Figure 2. Impact of AAVR nonsense variants on AAV-mediated transgene expression HeLa KIAA0319L−/− cells were transfected with AAVR expression plasmids (20 ng) encoding AAVR nonsense variants. Following a 48 h incubation, the cells were infected with AAV serotypes expressing NanoLuc (3 × 105 gc/cell). (A) Western analysis detection of wild-type AAVR (WT) or AAVR nonsense variant expression confirming absence of intact AAVR in cells transfected with nonsense variants. Representative of 4 replicates. (B) Quantification of western analyses. The level of AAVR expression was evaluated as a ratio to the level of expression of GAPDH. Mean ± SD plotted for each condition. (C–I) AAV-mediated transgene expression as a percentage of wild-type AAVR-mediated transgene expression. After transfection with AAVR nonsense variant or wild-type plasmid, the cells were transduced with different AAV serotypes expressing NanoLuc (3 × 105 AAV gc/cell). NanoLuc transgene expression from AAV vectors (serotypes 2, 5, 6, 8, 9, rh.10 and rh32.33) was assessed. Data from 3 replicate experiments normalized to WT are plotted (mean ± SEM). Asterisks indicate significance of difference compared to WT: ∗∗∗p < 0.001. Red bars…

Figure 3. Protein expression of the AAVR missense variants HeLa KIAA0319L−/− cells were transfected with 20 ng AAVR missense variant plasmid and incubated for 48 h. (A) Western analysis of wild-type AAVR (WT) or AAVR missense variant expression. Representative of 7 replicates. (B) Quantification of the western analyses. The level of AAVR expression was evaluated as a ratio to the level of expression of GAPDH and presented as a percentage of the ratio of wild-type AAVR to GAPDH in wild-type AAVR-transfected cells. The wild-type level is marked by the dotted line. Data represent quantification of 7 western analyses for each serotype (mean ± SD). Asterisks indicate significance of difference compared to WT: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 4. Effect of missense variants of human AAVR on AAV-mediated transgene expression mediated by different AAV serotypes (A–K) HeLa KIAA0319L−/− cells were transfected with 20 ng expression plasmid for each of the AAVR missense variants, incubated for 48 h, and then transduced with different AAV serotypes and incubated for an additional 72 h. Data from 5 replicate experiments normalized to wild type (WT) are plotted (mean ± SEM). Red bars indicate serotypes with transgene expression significantly reduced in the presence of an AAVR variant. Green bars indicate serotypes with transgene expression significantly increased in the presence of an AAVR variant. Black bars indicate serotypes with no significant change in transgene expression in the presence of an AAVR variant. Dotted line represents the mean transgene expression level mediated by wild-type AAVR transfection. Asterisks indicate significance of difference compared to WT: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 5. Assessment of potential mechanisms resulting in increased AAV-mediated transgene expression for AAV5 in the presence of AAVR variants (A) AAV5 binding analysis of wild-type AAVR (WT) or AAVR missense variant. Viral genome levels were quantified by qPCR targeting both NanoLuc transgene and RNase P gene, which served as a genomic reference standard. Data from 4 replicate experiments are plotted (mean ± SEM). (B) Nuclear localization of AAV genomes in cells expressing either wild-type AAVR (WT) or AAVR missense variant following AAV5 transduction. Viral genome levels were quantified by qPCR targeting both NanoLuc transgene and RNase P gene, which served as a genomic reference standard. Data from 3 replicate experiments are plotted (mean ± SEM). Asterisks indicate significance of difference compared to WT at the respective time point: ∗∗∗p < 0.001. Black bars represent 4-h time point, and red bars indicate 12-h time point.