Evaluation Of A New Antisense Oligonucleotide Chemistry-2’-O-Methoxyethvl In Duchenne Muscular Dystrophy

Objectives:Duchenne Muscular Dystrophy (DMD) is a lethal neuromuscular disorder caused by the absence of dystrophin protein, which encoded by the dystrophin gene. In our present study. we systemically evaluated a new antisense oligonucleotide (AO) chemistry-2"-O-Methoxyethyl (2-O-MOE) in the DMD disease model in vitro and in vivo, and wish to provide a candidate antisense oligonucleotide drug for treating DMD patients. Furthermore, we undertook the cellular uptake study with fluorescence-labeled MOE25(PS) AOs in H2K mdx cells, followed by co-localization experiments aiming to identify where MOEs majorly located. Subsequently we performed the tissue distribution study with fluorescence-labeled MOE AOs in adult mdx mice and tried to quantify the amount of MOE AOs in non-muscle and muscle tissues.Methods:1. In dose-dependent study, we chose300nM500nM and1μM2’-O-MOE modified AOs to transfect H2K max cells. RT-PCR technique was utilized to measure the efficiency of various AOs in inducing exon23skipping.2. In time-course study, we chose500nM as the tested concentration and two2’-O-MOE modified AOs were transfected into H2K mdx cells. After24h.48h.72h.96h post-transfection. RT-PCR was used to examine the exon23skipping efficiency.3. In order to measure the cytotoxicity of different2’-O-MOE modified AOs. cell viability experiments were carried out with the WST kit at higher concentrations.4. To compare with clinically tested2’OmePS AOs. three2’-O-MOE modified and2’OmePS AOs were transfected into H2K mdx cells at the concentration of500nM. After48h. RT-PCR was used for detecting the exon skipping efficiency.5. For the local intramuscular evaluation.5microgram selected2’-O-MOE AOs were injected into tibialis anterior (TA) muscles of adult mdx mice. Treated TA muscles were harvested two weeks post-injection and assayed by immunohistochemistry. RT-PCR and Western blot to measure the exon skipping efficiency and the level of dystrophin protein expression.6. Subsequently, we undertook systematic evaluation of the top candidate2’-O-MOE AOs-MOE25(PS) in adult mdx mice by administering these compounds intravenously, intraperitoneally and subcutaneously. Also different dosing regimes were investigated. All body-wide muscles were harvested and assayed as described above two weeks after the last injection.7. For cellular uptake study, we transfected H2K mdx cells with fluorescence-labeled MOE25(PS) AOs. followed by FACS analysis to quantify the efficiency of cellular uptake.8. For the co-localization experiment, laser scanning confocal fluorescence microscope was utilized for visualizing the localization of FITC-labeled MOE25(PS) AOs in H2K mdx cells after transfection.9. In the tissue distribution experiment. fluorescence-labeled MOE25(PS) AOs were injected into adult mdx mice intravenously at the dose of100mg/kg every other day for5times and all the body-wide tissues including liver. kidney and other muscle tissues were harvested and assayed by fluorescence plate-reader to quantify the compounds in various tissues.Results:1. With escalating doses, the exon skipping efficiency of3tested2’-O-MOE AOs increased accordingly, indicating a dose-dependent effect. Among them. MOE25(PS) AOs showed the highest activity with more than90%exon skipping detected at the concentration of500nM.2. In the time-course study. RT-PCR results demonstrated that48h time-point is the peak time in inducing exon23skipping for both MOE25(PO) and MOE25(PS) AOs.3. Cvtotoxicity assay showed that no toxic effect was observed for MOE25(PS) and MOE20(PS) AOs at higher concentrations (10μM). Whereas at the same concentration. for the MOE25(PO) AO. minor toxicity was detected.4. In order to compare the exon skipping efficiency with current clinically tested2’OmePS AOs.3different MOE and2’OmePS AOs were tested in H2K mdx cells. subsequent RT-PCR results indicated that the exon23skipping efficiency of MOE25(PS) AOs was significantly higher than those of2’OmePS and other tested MOE AOs at the concentration of500nM, suggesting that MOE25(PS) AO is likely a better candiate than2’OmePS.5. For the local intramuscular evaluation, all4tested AOs showed effective exon23skipping and restoration of dystrophin protein. Notably, there were uniform distribution of dystrophin-positive fibres throughout the muscle cross-section treated by MOE25(PS) AOs as shown by immunohistochemistry. RT-PCR and Western blot results indicated higher level of exon skipping and dystrophin expression in samples treated with MOE25(PS) AOs than those from other compounds.6. Subsequently the top candidate2’-0-MOE AOs-MOE25(PS) was evaluated systemically in adult mdx mice via different delivery routes and different dosing regimes. However, unexpectedly the immunostaining results only showed very few dystrophin-positive fibres in some skeletal muscles and no dystrophin-positive fibres were detected in heart. In line with the immunostaining results. RT-PCR only detected trace amount of exon skipping in some skeletal muscles.7. The following in vivo tissue distribution study indicated that the majority of FITC-labeled MOE25(PS) AOs entered kidney, liver and only trace amount of AOs was detected in muscles tissues.8. In the celluar uptake experiment, the fluorescence images showed that4h post-infection is the peak time for cellular uptake, corroborated by the following FACS result.9. For the co-localization experiment, confocal results demonstrated that most FITC-labeled MOE25(PS) AOs trapped in the cytoplasm and some located in lysosomes. And only a small portion of AOs were found in the nuclei.Conclusion:1.2’-O-MOE AOs are effective in inducing exon23skipping in H2K mdx cells and demonstrate the dose-dependent effect and also the peak time-point in inducing exon skipping is48h post-transfection. consistent with our previous report. There was no toxicitv detected for2’-O-MOE AOs at higher concentrations. 2.2’-O-MOE AOs show the potency in restoring the dystrophin protein expression from local intramuscular studies, but are ineffective in restoring dystrophin protein systemically.3. In vivo distribution study demonstrated that due to the lack of muscle-targeting property, large portion of MOE AOs entered kidnery, liver and only minor part of AOs detected in muscle tissues, providing evidences for the low exon skipping efficiency and the low level of dystrophin expression observed in systemic studies.4. Subsequent cellular uptake and co-localization studies indicated that most MOE AOs were trapped in the cytosolic compartment, implying that the release of AOs from cytoplasm will be crucial for improving the efficacy of MOE AOs in DMD.