Studies Of Novel Delivery Formulation F5 And Wild-type Mice In Duchenne Muscular Dystrophy

Objective: Duchenne muscular dystrophy(DMD) is an X-linked, lethal neuromuscular disorder caused by frame-disrupting mutations in the dystrophin gene. Currently there is no treatment available in clinic. Antisense oligonucleotide mediated exon-skipping therapeutics is one of the most promising approaches for treating DMD. However, recent phase III clinical trial failed to meet the primary endpoints, which is likely attributed to the insufficient systemic delivery as demonstrated in pre-clinical studies. This further underlined the importantance of developing safe, effective delivery technologies, compatible with the AOs development. Previously, we screened a number of different formulations in mdx mice intramuscularly and identified a few top candidates including F5, which can significantly enhance the potency of AOs. Based on previous studies, we tried to explore the potential of F5 in mdx mice systemically by comparing F5 with other four top candidates in the ability to enhance the activity of different AOs in mdx mice intramuscularly. Furthermore, we investigated the systemic effect of F5 on augmenting the activity of AOs and the potential drug-related toxicities in mdx mice via different dosing regimens. Our study will provide a new avenue and strategy for AO systemic delivery for DMD and serve as a cornerstone for clinical translation of F5. Second part of our study was to explore other potential DMD mouse models for accelerating the drug development for DMD given the accessibility and high maintainence cost of mdx mice.Methods:1. Based on previous work, we chose five top candidate formulations including F4, F5, F9, F10 and F12 for further evaluation in mdx mice. Five candidates were co-injected with 5μg MOE AOs into tibialis anterior(TA) muscles of mdx mice and TA muscles were harvested two weeks post-injection. Immunohistochemistry, RT-PCR and Western blot were used for examining the expression and distribution of dystrophin-positive fibers and the level of exon-skipping and dystrophin restoration.2. Five candidate formulations were co-injected with 5μg peptide nucleic acid(PNA) AOs into TA muscles of mdx mice and TA muscles were harvested two weeks post-injection. Immunohistochemistry, RT-PCR and Western blot were used for examining the expression and distribution of dystrophin-positive fibers and the level of exon-skipping and dystrophin restoration.3. Based on above results, F5 was chosen for further evaluation. F5 was co-injected with 2μg PMO, 2μg M12-PMO, 1μg B-MSP-PMO and 2μg R-PMO into TA muscles of mdx mice and TA muscles were harvested two weeks post-injection. Immunohistochemistry, RT-PCR and Western blot were used for examining the expression and distribution of dystrophin-positive fibers and the level of exon-skipping and dystrophin restoration.4. To determine the optimal concentration of F5, we tested 2.5% and 7.5% F5 in combination with PMO in mdx mice intramuscularly and compared with 5% F5, which is the concentration we used. Immunohistochemistry, RT-PCR and Western blot were used for examining the expression and distribution of dystrophin-positive fibers and the level of exon-skipping and dystrophin restoration.5. To systemically assess the enhancing effect of F5 in mdx mice, we applied a multiple low dosing regimen by injecting PMO at 25 mg/kg for 3 weekly injections. Body-wide muscle and liver, kidney were harvested two weeks after the last injection. The same methods for dectecting the dystrophin expression and exon-skipping were used as above. Meanwhile histological approaches were used for examining the potential drug-related toxicity.6. In order to examine how F5 influences the activity of PMO, we co-administered lissamine-labeled PMO with F5 in mdx mice intravenously at the dose of 25 mg/kg for 3 daily injections. Body-wide tissues were harvested four days after the last injection. IVIS small animal imaging system was utilized to evaluate the uptake and tissue distribution of lissamine-labeled PMO. Meanwhile, immunohistochemistry, RT-PCR and Western blot were used to examine the exon-skipping and dystrophin expression.7. To investigate the long-term efficacy of F5 in augmenting the activity of PMO, we co-administered F5 with PMO at the dose of 50 mg/kg with a combination of 3 weekly and 5 monthly repeated injections intravenously. Two weeks after the last injection, body-wide tissues were harvested for examining the level of exon-skipping and dystrophin expression with immunohistochemistry, RT-PCR and Western blot. Functional improvement was assessed with grip strength, relocalization of dystrophin-associated protein complex(DAPC) and measurement of biochemical parameters including creatine kinase(CK) and aspartate aminotransferase(AST), alanine aminotransferase(ALT). Routine H&E staining and immunohistochemistry for muscle and liver and kidney were performed to examine any potential adverse effect.8. To assess the feasibility of using wild-type mice as potential DMD anaimal models, we tested 6 different AO drugs in wild-type C57BL/6 mice intramuscularly. TA muscles were harvested 48 h after injection and assayed by RT-PCR to examine the level of exon-skipping.9. To test the duration of exon-skipping in C57BL/6 mice, we administered PMO, PNA and 2’Ome RNA AOs in mdx mice intramuscularly and harvested the tissues at different time-points including 48 h, 2-week and 4-week after injection. The exon-skipping was assayed by RT-PCR and compared with those of mdx mice.10. To determine whether there is any potential toxicity with AO administration, we applied H&E staining to examine the morphogical changes of treated TA muscles. 11. To examine the possibility of using other wild-type mice as DMD animal models, we tested PMO, PNA and 2’Ome RNA AOs in ICR, C3 H and BABL/C mice. RT-PCR was used to evaluate the exon-skipping efficiency and compared with that of mdx mice to verify the viability of other wild-type mice as animal models for DMD drug screening.12. To investigate the possibility of using wild-type mice as DMD animal models for systemic drug screening, we administered PMO at the dose of 25 mg/kg for 3 weekly injections into C57BL/6 and C3 H mice intravenously. Two weeks after last injection, body-wide muscles were harvested and examined for the exon-skipping efficiency with RT-PCR.Results:1. The results of local intramuscular injection of F4, F5, F9, F10 and F12 with MOE in mdx mice revealed that all the tested formulations improved the exon-skipping efficiency and dystrophin expression compared to saline. In particular, F5 and F12 performed better than others and the level of exon-skipping was 3.53-fold and 3.45-fold higher than that of saline, respectively.2. The combinatorial test of F4, F5, F9, F10 and F12 with PNA in mdx mice intramuscularly indicated that all the tested formulations increased the exon-skipping efficiency and level of dystrophin expression. In comparison with saline, approximately 1.27 to 1.54-folds increase. There was no significant difference between formulations. Taken together, our local data indicated that F5 can significantly augment the activity of different AOs.3. Further evaluation of F5 with other AOs including M12-PMO, B-MSP-PMO and R-PMO intramuscularly demonstrated that F5 can enhance the activity of a variety of AOs. Notably, the highest enhancement was achieved with the combination of PMO and F5, approximately 4.24 fold.4. The concentration test revealed that 5% is the optimal concentration for F5 to enhance the activity of PMO compared to 2.5% and 7.5%.5. Systemic evaluation of F5 with PMO in mdx mice indicated that F5 can significantly augment the activity of PMO in inducing exon-skipping and dystrophin restoration. Histological examination revealed no drug-related toxicity.6. The mechanistic study indicated a significant increase in the cellular uptake of lissamine-labeled PMO in muscles treated with F5 and lissamine-labeld PMO compared to saline, suggesting the enhanced delivery accounts for the increased activity of PMO in mdx mice.7. Long-term study of F5 and PMO in mdx mice demonstrated that long-term repeated administration of F5 and PMO resulted in improvement in the level of exon-skipping and dystrophin restoration compared to untreated age-matched mdx controls. However there is no difference between F5 and saline. And repeated treatments with PMO contributed to the functional and phenotypic rescue of mdx mice without any detectable toxicity, though there is no difference between F5 and saline, indicating no beneificial effect can be achieved with long-term repeated injections of F5.8. Local evaluation of 6 different AOs in wil-type C57BL/6 mice demonstrated that comparable level of exon-skipping was achieved between C57BL/6 and mdx mice for all tested AOs, suggesting C57BL/6 mice can be a viable model for DMD drug screening.9. The duration study of PMO, PNA and 2’Ome RNA AOs in C57BL/6 mice revealed that different exon-skipping efficiency can be yielded at different time-points with the same AO chemistry. The exon-skipping efficiency reached peak at 4 weeks after injection with PMO and PNA, whereas the peak expression for 2’Ome RNA AOs was 48 h post-injection. Overall, exon-skipping can be detectable at 48 h after injection for all tested AOs. Further histological examination displayed no drug-related adverse effect.10. Local evaluation of PMO, PNA and 2’Ome RNA AOs in other wild-type mice including C3 H, BALB/C and ICR indicated that detectable level of exon-skipping was achieved with 3 tested AOs in 3 different wild-type mice but the efficiency varies between different strains of wild-type mice. Notably, levels of exon-skipping obtained in C57BL/6 and C3 H and mdx mice were most closely matched, followed by ICR and BALB/C mice.11. Systemic validation in C57BL/6 and C3 H revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice.Conclusions:1. Local evaluation of 5 top candidate formulations with 6 different AOs in mdx mice demonstrated that 5 top candidate formulations can significantly increase the activity of AOs, though to different extents, compared to saline; and the highest activity was achieved with the combination of F5 and PMO.2. 5% F5 is the optimal concentration to use.3. Systemic investigation of F5 indicated that beneificial effect can be established with short-term repeated administration of F5 but to a less extent with long-term repeated administration of F5.4. The mechanistic study elucidated that the enhanced delivery contributes to the increased activity of PMO in mdx mice.5. Local and systemic evaluation of wild-type mice demonstrated that wild-type mice can be a viable and practicle animal model for DMD drug screening though the systemic sensitivity to AOs is less than that of mdx mice.