Figure 2. Tricistronic shRNA delivered in vivo by AAV vectors efficiently downregulates endogenous PABPN1 without affecting optPABPN1 expression in vivo.

Eighteen weeks after intramuscular AAV injections, TA muscles were collected and expression of PABPN1 was analysed. (a) qRT-PCR analysis of PABPN1 mRNA normalized to the housekeeping gene RplP0 mRNA shows a statistically significant decrease in PABPN1 expression in muscles injected with shRNA3X and optPABPN1 compared with muscles injected with saline. (b) Western blot for PABPN1 expression shows that the treatments with AAV-shRNA3X alone or in combination with AAV-optPABPN1 significantly knock down the endogenous PABPN1. (c) Densitometric analysis of western blot for PABPN1 detection shows statistically significant reduction in protein expression when muscles are treated with shRNA3X (with or without optPABPN1 expression) compared to the level of A17 muscles. PABPN1 expression was normalized to the relative vinculin expression. (d) qRT-PCR analysis for optPABPN1 mRNA normalized to the housekeeping gene Rplp0 mRNA shows significant expression in muscles where AAV-optPABPN1 was injected (with or without shRNA3X co-expression). (e) Representative western blot for MYC-tag shows that the epitope is detected in all muscles treated with AAV-optPABPN1 alone or in combination with AAV-shRNA3X. The arrow shows the band detected at the correct molecular weight. (f) Averages obtained by densitometric analysis of MYC-tag detected in samples treated with AAV-optPABN1 only or with both AAV-shRNA3X and optPABPN1 viral vectors indicates that shRNA3X does not affect optPABPN1 protein amount when co-expressed in muscles injected with both vectors. Two-tailed Student t-test, n=8, NS, not significant. (g) Detection of PABPN1 inclusions (green) and Laminin (red) by immunofluorescence in sections of treated muscles. Sections were pre-treated with 1 M KCl to discard all soluble PABPN1 from the tissue. Nuclei are counterstained with DAPI (blue). Bar, 200 μm. (h) Quantification of percentage of nuclei containing INIs in muscle sections indicates that treatments with either AAV-shRNA3X or both AAV-shRNA3X and optPABPN1 significantly reduce the amount of INIs to about 10% and 5%, respectively, compared to saline-injected A17. For a,c,d,h, n=6 (saline-treated A17 or FvB muscles) or n=8 (all the other groups). Data are presented as mean±s.e.m. One-way ANOVA test with Bonferroni post-doc test **P<0.01, ***P<0.001, NS, not significant.