RNA-Based Therapy Utilizing Oculopharyngeal Muscular Dystrophy Transcript Knockdown and Replacement

Abstract

Oculopharyngeal muscular dystrophy (OPMD) is caused by a small expansion of a short polyalanine (polyAla) tract in the poly(A)-binding protein nuclear 1 protein (PABPN1). Despite the monogenic nature of OPMD, no treatment is currently available. Here we report an RNA replacement strategy that has therapeutic potential in cell and C. elegans OPMD models. We develop selective microRNAs (miRNAs) against PABPN1, and we report that miRNAs and our previously developed hammerhead ribozymes (hhRzs) are capable of reducing the expression of both the mRNA and protein levels of PABPN1 by as much as 90%. Since OPMD derives from a very small expansion of GCG within the polyAla tract, our hhRz and miRNA molecules cannot distinguish between the wild-type and mutant mRNAs of PABPN1. Therefore, we designed an optimized-codon wild-type PABPN1 (opt-PABPN1) that is resistant to cleavage by hhRzs and miRNAs. Co-expression of opt-PABPN1 with either our hhRzs or miRNAs restored the level of PABPN1, concomitantly with a reduction in expanded PABPN1-associated cell death in a stable C2C12 OPMD model. Interestingly, knockdown of the PABPN1 by selective hhRzs in the C. elegans OPMD model significantly improved the motility of the PABPN1-13Ala worms. Taken together, RNA replacement therapy represents an exciting approach for OPMD treatment.

Keywords: OPMD; PABPN1; RNA replacement therapy; microRNAs; polyalanine disorders; ribozymes.

Graphical abstract

Figure 1

hhRzs and miRNAs Target Different PABPN1 Domains A schematic representation of human PABPN1 protein showing several domains: polyalanine stretch, proline-rich region, coiled-coil domain, oligomerization domains, and two RNA-binding domains. The figure shows the exact location of each hhRz and miRNAs targeting the different domains of PABPN1.

Figure 2

hhRzs Are Effective in In Vivo Transgenic OPMD C. elegans (A) Western blot showing the high efficiency of hhRzs in knocking down PABPN1 protein level in OPMD C. elegans. PABPN1-13Ala and PABPN1-0Ala animals were crossed with hhRz144, hhRz363, or hhRz437. The crossed animals were then genotyped before protein extraction. The protein levels of PABPN1 were markedly reduced in PABPN1-13 animals crossed with any one of the three hhRzs compared to non-crossed animals. This decrease was not obvious in PABPN1-0Ala animals crossed with any one of the three hhRzs. The immunoblot was performed using GFP antibody. Actin antibody was used to indicate equal loading across all lanes. (B) hhRzs lead to a decrease in GFP signal when crossed with PABPN1-13A animals. The top image shows an adult C. elegans expressing myo-3::GFP; myo-3::PABPN1-13A in the nuclei of body-wall muscle cells. The worms in the three images below co-express myo-3::GFP; myo-3::PABPN1-13A with hhRz144, hhRz363, or hhRz437. Expression of hhRz causes fading of the GFP signal, indicating silencing of PABPN1. Scale bar, 1 mm. (C) Knockdown of the PABPN1 alleviates the adult-stage abnormalities in the C. elegans OPMD model. The motility of mutant animals expressing 13Ala human PABPN1 protein declined quickly, such that these animals were completely paralyzed. However, a significant improvement in motility was observed in PABPN1-13Ala animals crossed with hhRz144, hhRz363, or hhRz437 compared to non-crossed PABPN1-13Ala animals throughout all time points, reaching the motility level detected in control animals (PABPN1-0Ala) (*p < 0.0001). Motility recordings were generated using a Microtracker device, an automated tracking system that detects the animal movement through infrared microbeam light scattering. Simply, each microtiter well is crossed by at least one infrared microbeam, scanned more than 10 times/s. The detected signal is then digitally processed to register the amount of animal movement in a fixed period of time. At the indicated time points, the number of movements was scored at 22°C, for 10 consecutive h.

Figure 3

The Effects of hhRz and miRNA Delivery with and without opt-PABPN1 in HeLa Cells (A) Percentage of cell survival was determined by live-stage microscopy in cells transfected with RNA molecules with or without opt-PABPN1. Co-transfection of opt-PABPN1 (right side of the graph) with hhRzs or miRNAs shows a striking increased percentage of cell survival compared to cells transfected with only hhRzs or miRNAs at different time points (mean ± SE; *p < 0.001 versus nontreated samples, ANOVA). (B) Measurement of endogenous PABPN1 gene expression in the HeLa cells transfected with one hhRz, a combination of two hhRzs, or one miRNA by qPCR. Controls were also included. RNA was collected 48 h post-transfection. qPCR analysis evaluating the endogenous PABPN1 mRNA levels showed a significant downregulation of PABPN1 for cells transfected with hhRz144, hhRz363, and hhRz437 with respect to the nonsense hhRz. A substantial reduction of PABPN1 mRNA levels was detected when HeLa cells were co-transfected with the corresponding two hhRzs. RNA polymerase II served as a normalization control gene. Values are given as means ± SE of three independent experiments and each experiment was performed in triplicate. *p < 0.005, n = 3. Significant downregulation of human endogenous PABPN1 mRNA was observed after transfecting HeLa cells with the six different miRNAs targeting PABPN1. *p < 0.005, n = 3. The figure shows a clear increase in PABPN1 mRNA levels when opt-PABPN1 was introduced into cells with the hhRzs or miRNAs, indicating that opt-PABPN1 is capable of enhancing PABPN1 mRNA levels (right side of the graph). (C) ELISA results for PABPN1 show a clear inhibition of endogenous PABPN1 protein following hhRz or miRNA transfection. This inhibition was clearly rescued by co-expressing opt-PABPN1 with hhRzs or miRNAs. Protein levels were measured in supernatants of HeLa 48 h post-transfection. The values represent the means ± SDs from three independent experiments performed in triplicate. *p < 0.05 using Student’s t test.

Figure 4

The Effects of hhRz and miRNA Delivery with and without opt-PABPN1 in HEK239T Cells (A) Percentage of cell survival was determined by live-stage microscopy in cells transfected with RNA molecules with or without opt-PABPN1. Co-transfection of opt-PABPN1 (right side of the graph) with hhRzs or miRNAs shows a striking increased percentage of cell survival compared to cells transfected with hhRzs or miRNAs but without opt-PABPN1 at different time points (mean ± SE; *p < 0.001 versus nontreated samples, ANOVA). (B) Measurement of endogenous PABPN1 gene expression in the HEK239T cells transfected with individual hhRz, a combination of two hhRzs, or individual miRNA by qPCR. Controls were also included. RNA was collected 48 h post-transfection. qPCR analysis evaluating the endogenous PABPN1 mRNA levels showed a significant downregulation of PABPN1 for cells transfected with hhRz144, hhRz363, and hhRz437 with respect to the nonsense hhRz. A substantial reduction of PABPN1 mRNA levels was detected when HEK239T cells were co-transfected with the corresponding two hhRzs. RNA polymerase II served as a normalization control gene. Values are given as means ± SE of three independent experiments and each experiment was performed in triplicate. *p < 0.005, n = 3. Significant downregulation of human endogenous PABPN1 mRNA was observed after transfecting HEK239T cells with the six different miRNAs targeting PABPN1. *p < 0.005, n = 3. The figure shows a clear increase in PABPN1 mRNA levels when opt-PABPN1 was co-transfected with RNA molecules, indicating that opt-PABPN1 is capable of enhancing PABPN1 mRNA levels (right side of the graph). (C) ELISA results for PABPN1 show a clear inhibition of endogenous PABPN1 protein following hhRz or miRNA transfection. This inhibition was clearly rescued by co-expressing opt-PABPN1 with hhRzs or miRNAs. Protein levels were measured in supernatants of HEK239T 48 h post-transfection. The values represent the means ± SDs from three independent experiments performed in triplicate. *p < 0.05 using Student’s t test.

Figure 5

The Effects of hhRz and miRNA Delivery with and without opt-PABPN1 in C2C12 (A) Percentage of cell survival was determined by live-stage microscopy in cells transfected with RNA molecules with or without opt-PABPN1. Co-transfection of opt-PABPN1 (right side of the graph) with hhRzs or miRNAs shows a striking increased percentage of cell survival compared to cells transfected with hhRzs or miRNAs but without opt-PABPN1 at different time points (mean ± SE; *p < 0.001 versus nontreated samples, ANOVA). (B) Measurement of endogenous PABPN1 gene expression in the C2C12 cells transfected with individual hhRz, a combination of two hhRzs, or individual miRNA by qPCR. Controls were also included. RNA was collected 48 h post-transfection. qPCR analysis evaluating the endogenous PABPN1 mRNA levels showed a significant downregulation of PABPN1 for cells transfected with hhRz144, hhRz363, and hhRz437 with respect to the nonsense hhRz. A substantial reduction of PABPN1 mRNA levels was detected when C2C12 cells were co-transfected with the corresponding two hhRzs. RNA polymerase II served as a normalization control gene. Values are given as means ± SE of three independent experiments and each experiment was performed in triplicate. *p < 0.005, n = 3. Significant downregulation of human endogenous PABPN1 mRNA was observed after transfecting C2C12 cells with the six different miRNAs targeting PABPN1. *p < 0.005, n = 3. The figure shows a clear increase in PABPN1 mRNA levels when opt-PABPN1 was co-transfected with hhRzs or miRNAs, indicating that opt-PABPN1 is capable of enhancing PABPN1 mRNA levels (right side of the graph). (C) ELISA results for PABPN1 show a clear inhibition of endogenous PABPN1 protein following hhRz or miRNA transfection. This inhibition was clearly rescued by co-expressing opt-PABPN1 with these RNA molecules. Protein levels were measured in supernatants of C2C12 48 h post-transfection. The values represent the means ± SDs from three independent experiments performed in triplicate. *p < 0.05 using Student’s t test.

Figure 6

Developing the opt-PABPN1 that Is Resistant to Knockdown by the hhRzs and miRNAs (A) Subcellular localization of wild-type PABPN1. C2C12 cells were transfected with GFP-wild-type (WT)-PABPN1-10Ala and visualized under the fluorescent microscope 48 h post-transfection. Wild-type PABPN1 is a nuclear protein with widespread staining in the nucleoplasm. It is mostly concentrated in discrete nuclear domains called speckles. Phase images are also shown. (B) Cellular expression of opt-PABPN1 is similar to wild-type PABPN1-10Ala. Fluorescent microscopic images show the subcellular expression of opt-PABPN1-Dsred. C2C12 cells were transiently transfected with the opt-PABPN1-Dsred. Images were captured 48 h post-transfection using a fluorescent microscope. Scale bars are indicated. (C) opt-PABPN1 enhances protein expression. As shown by western blot, there was a clear increase in the level of PABPN1 expression after opt-PABPN1 transfection. The level of PABPN1 expression is higher in lane 1 (opt-PABPN1-GFP) and in lane 3 (opt-PABPN1-Dsred) compared to lane 2 (non-transfected cells). The proteins were then extracted at 48 h after transfection. PABPN1 antibody was used to measure PABPN1 protein level. The actin expression is used to indicate equal loading across all lanes.

Figure 7

The Effects of hhRz and miRNA Delivery with and without opt-PABPN1 in a Stable C2C12 OPMD Cell Model (A) Co-transfection of opt-PABPN1 with hhRzs increases the cell survival in C2C12-17Ala myoblasts. Representative images show C2C12-17Ala myoblasts treated with hhRzs with or without opt-PABPN1. PABPN1 knockdown by hhRzs induced significant cell death in C2C12-17Ala myoblasts (left), but co-expressing opt-PABPN1 with hhRzs rescued cells from cell death (right). Cells were monitored using a live-stage microscope every day and up to 14 days in culture. Images were captured at day 10. Scale bar, 100 μM. (B) Co-transfection of opt-PABPN1 with miRNAs increases the cell survival in C2C12-17Ala myoblasts. PABPN1 knockdown by miRNAs induces significant cell death in C2C12-17Ala myoblasts (left). Co-expressing opt-PABPN1 with miRNAs rescued cells from cell death (right). Cells were monitored using a live-stage microscope every day and up to 14 days in culture. Images were captured at day 10. Scale bar, 100 μM. (C) Percentage of cell survival was determined by live-stage microscopy. C2C12-17Ala myoblasts were transfected with hhRzs or miRNAs with or without opt-PABPN1 and monitored at different days in culture. In C2C12-17Ala myoblasts, the cell death started at day 7 of culture and reached its highest point after day 10. Co-transfection of opt-PABPN1 (right side of the graph) with hhRzs or miRNAs shows a striking increased percentage of cell survival compared to cells transfected with only hhRzs or miRNAs at days 7 and 10 in culture (mean ± SE; *p < 0.001 versus nontreated samples, ANOVA). It is important to mention that expressing opt-PABPN1 alone in cell was not enough to protect against the cell death associated with _expPABPN1-17Ala. (D) Measurement of endogenous PABPN1 gene expression in the C2C12-17Ala myoblasts transfected with one hhRz, a combination of two hhRzs, or one miRNA by qPCR. Controls were also included. RNA was collected 4 days post-transfection. qPCR analysis evaluating the endogenous PABPN1 mRNA levels showed a significant downregulation of PABPN1 for cells transfected with hhRz144, hhRz363, and hhRz437, with respect to the nonsense hhRz. A substantial reduction of PABPN1 mRNA levels was detected when C2C12-17Ala myoblasts were co-transfected with the corresponding two hhRzs. RNA polymerase II served as a normalization control gene. Values are given as means ± SE of three independent experiments and each experiment was performed in triplicate. *p < 0.005, n = 3. Significant downregulation of human endogenous PABPN1 mRNA was observed after transfecting C2C12-17Ala myoblasts with the six different miRNAs targeting PABPN1. *p < 0.005, n = 3. The figure shows a clear increase in PABPN1 mRNA levels when opt-PABPN1 was introduced into cells with the hhRzs or miRNAs, indicating that opt-PABPN1 is capable of enhancing PABPN1 mRNA levels (right side of the graph). (E) ELISA results for PABPN1 show a clear inhibition of endogenous PABPN1 protein following hhRz or miRNA transfection. This inhibition was clearly rescued by co-expressing opt-PABPN1 with hhRzs or miRNAs. Protein levels were measured in supernatants of C2C12-17Ala myoblasts 4 days post-transfection. The values represent the means ± SDs from three independent experiments performed in triplicate. *p < 0.05 using Student’s t test.