PABPN1 gene therapy for oculopharyngeal muscular dystrophy

Abstract

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment.

Figure 1. The tricistronic construct efficiently silences endogenous normal and expanded PABPN1 without affecting optPABPN1 expression in vitro.

(a) Three constructs were cloned into pAAV vectors and used in this study: a tricistronic shRNA construct including hairpins sh-1, sh-2 and sh-3 driven each by a different polymerase III promoter (U61, U69 and H1, respectively), the sequence-optimized PABPN1 (optPABPN1) driven by the SPc5-12 promoter and tagged with a MYC-tag, and the human expanded PABPN1 (expPABPN1) driven by the SPc5-12 promoter and tagged with a FLAG-tag. (b) HEK293T cells were transfected with 4 μg per well of pAAV-shRNA3X in triplicate with or without AAV plasmids expressing expPABPN1 or optPABPN1. Untransfected cells and cells transfected with AAV plasmid expressing shRNA for HBVpol were used as a control. Seventy-two hours post transfection, samples were collected and PABPN1 was detected by western blot and quantified by densitometric analysis using ImageJ software. Transfection was performed twice. PABPN1 expression in each condition was normalized by GAPDH expression level and then by the value of untransfected cells. Transfection with a plasmid expressing shRNA3X induced efficient PABPN1 knockdown even when expPABPN1 was co-expressed compared to untransfected cells. The co-expression with optPABPN1 restored PABPN1 expression to the normal level. (c) Representative image of a western blot used for these analyses. (d) FLAG is not detected when samples are transfected with shRNA3X. However, in samples prepared from cells transfected with pAAV-opPABPN1, MYC-Tag is detected by western blot. Detection of MYC-Tag shows that optPABPN1 is resistant to degradation by shRNA3X in vitro. Data are presented as mean±s.e.m., n=4 (shRNA3X+optPABPN1) or n=6 (all the other groups). One-way ANOVA test with Bonferroni post-hoc test *P<0.05, **P<0.01, NS, not significant.

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.

Figure 3. In vivo PABPN1 depletion induces muscle degeneration that can be prevented by co-optPABPN1 expression.

(a) H&E staining of sections from treated muscles of 30-week-old mice indicates that depleting endogenous PABPN1 in A17 muscles increases the amount of centrally nucleated fibres. Bar, 200 μm. (b) Immunostaining for neonatal MHC (green), laminin (red) and DAPI (blue) shows small regenerating neonatal MHC-positive myofibres in muscles treated with shRNA3X only. Bar, 200 μm. (c) Quantification of centrally nucleated fibres indicates that co-injecting AAV-optPABPN1 preserves the amount of fibres with central nuclei to the same level observed in saline-injected A17 muscles, thus preventing muscle degeneration. (d) qRT-PCR analysis of Myh3 mRNA encoding embryonic MHC normalized to the housekeeping gene RplP0 mRNA confirms the regeneration process in shRNA3X-treated muscles. (e) H&E staining of sections from treated muscles shows that depleting endogenous PABPN1 in WT muscles increases the amount of centrally nucleated fibres. Bar, 200 μm. (f) Quantification of centrally nucleated fibres in treated muscles indicates that PABPN1 depletion in WT muscles induce muscle degeneration, as for A17 treated muscles. (g) qRT-PCR analysis of Myh3 mRNA encoding embryonic MHC normalized to the housekeeping gene RplP0 mRNA shows that a regeneration process is ongoing in shRNA3X treated muscles. (h) PABPN1 depletion in WT muscles reduces muscle weight compared to contralateral muscles. (i,j) PABPN1 inhibition decreases both absolute maximal tetanic and specific maximal force generated by TA muscles of wild-type mice. Data are presented as mean±s.e.m. For c,d, one-way ANOVA test with Bonferroni post-hoc test, n=6 (saline-treated A17 or FvB muscles) or n=8 (all the other groups). For f-j, two-tailed Student t-test, n=6, *P<0.05, ***P<0.001, NS, not significant.

Figure 4. In vivo co-administration of AAV-shRNA3X and AAV-optPABPN1 diminishes muscle fibrosis and myofibre atrophy and improves the functionality of treated muscles.

(a,b) Morphometric evaluation of Sirius red staining (a) or collagen VI immunostaining (b) in treated muscles of 30-week-old mice shows a significant reduction in fibrosis in muscles treated with AAVs expressing a combination of shRNA3X and optPABPN1 compared to saline-treated muscles. (c) The average of myofibre sizes per group shows that myofibres of muscles treated with AAV-optPABPN1 only or in combination with AAV-shRNA3X are larger than myofibres of muscles treated with saline. (d) Eighteen weeks after AAVs injection no difference was detected in the muscle weight of AAV-treated muscles compared to saline-injected TA of A17 mice. (e) Maximal force generated by TA muscles of treated mice was measured by in situ muscle physiology, co-injecting AAV-shRNA3X and AAV-optPABPN1 significantly increased the absolute maximal tetanic force generated by TA muscles. (f) Normalization of maximal force by muscle weight provides a measure of the muscle strength per unit of skeletal muscle called specific maximal force. The co-injection of the two AAVs normalized the specific maximal force of TA muscles to the level detected by wild-type muscles. Data are represented as mean±s.e.m. a–c: n=6 (saline-treated A17 or FvB muscles) or n=8 (all the other groups). d–f: n=8 (saline-treated A17 or FvB muscles) or n=10 (all the other groups). One-way ANOVA test with Bonferroni post-hoc test, *P<0.05, ***P<0.001, NS, not significant.

Figure 5. Transcriptome of A17 muscles treated with the combined approach is normalized to the one of WT mice.

(a) Principal component analysis of microarray data shows wide segregation of 30-week-old A17 mice clusters (in red) away from wild-type FvB mice clusters (in blue). Muscles treated with combined AAVs (shRNA3X and optPABPN1, in dark green) clustered very close to wild-type mice, while muscles treated with shRNA3X only (in light blue) clustered closer to FvB muscles than optPABPN1-injected muscles (light green). (b,c) Fold change in log2 (log2FC) of upregulated and downregulated genes in A17-treated groups compared to FvB. The treatments (A17 saline versus FvB, optPABPN1 versus FvB, shRNA3X versus FvB or shRNA3X+optPABPN1 versus FvB) are indicated under the graph. In A17 saline compared to FvB, 452 and 413 genes were up- and downregulated respectively. Ninety-eight per cent of these dysregulated (either up- or downregulated) transcripts showed a complete return to normal expression after the gene therapy treatment. n=3 (A17 and FvB injected with saline and A17 injected with optPABPN1) or n=4 (A17 injected with shRNA3X and A17 injected with shRNA3X+optPABPN1).

Figure 6. PABPN1 inhibition in human OPMD myoblasts induces cell death that can be prevented by optPABPN1 co-expression.

(a,b) Primary OPMD myoblast cells were transduced with shRNA3X tricistronic cassette, shRNA-HBVpol, shRNA3X-optPABPN1 or shRNA HBVpol-optPABPN1 LV vectors. GFP observed 4 and 10 days post transduction demonstrates that no GFP+ cells survived on the condition transduced with shRNA3X. However, co-transduction with shRNA-HBVpol-optPABPN1 prevented cell death. Bar, 100 μm.

Figure 7. The combined treatment with AAVs abrogates INIs and restores normal PABPN1 expression improving both the gain of toxic function (GF) and the loss of physiological function (LF) mechanisms of the disease in the A17 mouse model.

(a) In wild-type FvB skeletal muscle, the endogenous wild-type PABPN1 (green) moves to the nucleus where it can function normally. The amount of normal PABPN1 that achieves the threshold for the optimal cell survival and functionality as shown in the graph in the upper left side. There is no loss of function (LF) or gain of function (GF), labelled with N (Not). (b) In A17 mice the expression of expPABPN1 (red) induces aggregate formation where normal PABPN1 and several other proteins as well as polyA RNAs are trapped. This might respectively induce both a loss of function (LF) associated to a minor amount of normal PABPN1 available and a toxic gain of function (GF) due to the sequestration of other macromolecules associated to dysregulated biological pathways, labelled as Y (Yes). (c) After injection of AAV-optPABPN1, the newly expressed optPABPN1 (blue) counteracts the lack of available normal PABPN1 (that is, it may reduce the loss of function mechanism of the disease) but it does not affect aggregate formation nor the toxic gain of function mechanism. No improvement of the pathology is observed. (d) By delivering ddRNAi to knockdown endogenous PABPN1, and after its conversion to siRNA by DICER and RISC complexes, aggregates are abrogated and this is likely associated to a reduction on the gain-of-function mechanism. However the normal PABPN1 downregulation promotes muscle degeneration. Again, no improvement of the pathology is observed. (e) Only the co-injection of the two vectors inhibits the aggregate formation while rescuing the normal PABPN1 to the level vital for optimal cell survival and functionality in A17 muscles (suggesting that both the toxic gain of function and the loss of function effect might be significantly reduced).