Investigation Of Peptide Nucleic Acid In Duchenne Muscular Dystrophy Disease Model

Objectives Duchenne muscular dystrophy(DMD) is an X-linked fatal muscle degenerative disorder that arises from frame-disrupting mutations in the dystrophin gene, without any effective treatment available in clinic. Antisense oligonucleotides(AOs) mediated exon-skipping therapeutics shows huge potential in DMD treatment. Despite the promising results from phase II clinical trials with two lead drugs(2’Ome RNA and PMO), the recent failure of 2’Ome RNA to meet the primary endpoints from phase III trials and the extensional studies with PMO required by FDA further underline the importance of developing other effective candidate AO drugs for DMD. Peptide nucleic acid(PNA) is a recently-developed synthetic chemistry, bearing unique properties including high affinity to target sequence, high specificity and being neutral. We demonstrated the potential of PNAAOs in inducing exon-skipping in mdx mice intramuscularly in our previous study. However its systemic potential remains to be explored. In this study, we further extend the length of PNA AOs to the similar lengths of PMO used in current clinical trials. Evaluation of longer PNA AOs in mdx mice intramuscularly revealed that there is an evident length-dependent effect, with30 mer showing superiority to other shorter versions. However, subsequent systemic tests with longer PNA AOs indicated a strong acidity with longer PNA AOs,particularly with 30 mer PNA AOs showing stronger acidity than 25 and 20 mer PNA AOs, displaying a length-dependent acidity response, which precluded further investigation of PNA30 AOs and PNA25 AOs in mdx mice systemically at higher doses. In contrast, PNA20 AOs shows the best combination of activity, solubility and safety based on current PNA AOs synthesis technology. Systemic evaluation and optimization of dosing regimens of PNA20 AOs in mdx mice revealed that functional level of dystrophin can be restored with PNA20 AOs at the dose of 100 mg/kg, five weekly repeated injections. Taken altogether, our study showed that PNA AOs can induce effective exon-skipping and dystrophin restoration at 100 mg/kg for 5 weekly repeated injections in mdx mice, resulting in functional improvement without any detectable toxicity, demonstrating the potential of PNA AOs as an alternativechemistry for DMD treatment.Methods:1. Optimization of different lengths of PNA AOs in mdx mice intramuscularly.Different lengths of PNA AOs were designed including PNA20(+2-18), PNA25(+7-18), PNA26(+10-16), PNA28(+7-21), PNA30(+7-23) and PNA30(+14-16)and were injected into tibialis anterior( TA) muscles of mdx mice, respectively.Treated TA muscles were harvested two weeks after injection and assayed by immunohistochemistry, RT-PCR and Western blot to measure the exon 23 skipping efficiency and the restoration of dystrophin protein expression.2. Systemic evaluation of candidate PNA AOs in mdx mice. Based on local intramuscular studies as described above, two top candidate PNA AOs(PNA25and PNA30) were tested in mdx mice intravenously at the dose of 25 mg/kg for 3weekly injections. Body-wide peripheral muscle groups were harvested four weeks after injection and assayed by immunohistochemistry, RT-PCR and Western blot to measure the exon 23 skipping efficiency and the restoration of dystrophin protein expression.3. Impact of the solution acidity on the solubility and activity of PNA AOs. From systemic studies mentioned above, we noticed the high acidity of PNA AO solution, particularly with PNA30 and PNA25 AOs. Subsequently, we adjusted the p H value of PNA25 AOs solution with 0.1M Na OH to make p H=2, 3, 4, 5, 6and 7 PNA solution, respectively. The concentration was measured by Nanodrop.PNA25 AOs at different p H conditions were injected into mdx TA muscles,respectively. Treated TA muscles were harvested two weeks after injection and assayed by immunohistochemistry, RT-PCR and Western blot to measure the exon23 skipping efficiency and the restoration of dystrophin protein expression.Meanwhile, p H<1,=2-3 and p H=4-5 PNA25 AO solution were evaluated in mdx mice intravenously at 25 mg/kg dose for 3 weekly injections, respectively.Body-wide muscles were harvested four weeks after last injection and assayed by immunohistochemistry to measure the expression of dystrophin protein.4. Systemic optimization of different lengths of PNA AOs in mdx mice. PNA25 and PNA20 AOs were administered into mdx mice intravenously at the dose of 50mg/kg for 3 weeks at weekly interval. Body-wide muscles were harvested four weeks after last injection and assayed by immunohistochemistry, RT-PCR and Western blot to measure the exon 23 skipping efficiency and the restoration of dystrophin protein expression. Meanwhile, we also compared PNA20 to PNA18 AOs, which showed efficient exon-skipping activity in our previous local intramuscular study, in inducing exon-skipping and dystrophin restoration in mdx mice intravenously at the dose of 100 mg/kg for 3 weeks at weekly interval.5. Systemic evaluation PNA20 AOs in mdx mice. To further explore the potential of PNA20 AOs, single, triple and five repeated injections of PNA20 AOs were undertaken intravenously at the dose of 100 mg/kg at weekly interval, respectively.Body-wide tissues were harvested two weeks after last injection and assayed by immunohistochemistry, RT-PCR and Western blot to measure the exon-skipping efficiency and the restoration of dystrophin protein. Grip strength, re-localization of dystrophin-associated protein complex(DAPC) and creatine kinase(CK) were examined to assess the functional rescue. To determine the potential drug-related side effects, we performed histological H&E staining of liver and kidney tissue sections and examined biochemical parameters including AST( Aspartate transaminase), ALT(Alanine aminotransferase) and the presence of CD3+ and CD68+ T lymphocytes in diaphragmatic sections of mdx mice treated with PNA20 AOs.Result:1. Optimization of different lengths of PNA AOs in mdx mice intramuscularly showed that PNA20, PNA25 and PNA30 AOs elicited effective exon-skipping and dystrophin restoration in mdx mice, with PNA30 exhibiting significantly higher activity than other AOs. Only second to PNA30, PNA25 AOs also demonstrated higher potency than other AOs, indicating a length-dependent effect.2. As PNA30,PNA25 and PNA20 AOs showed significantly higher activity than other AOs, we first tested PNA30 and PNA25 AOs in mdx mice intravenously at the dose of 25 mg/kg for 3 weekly injections. The results revealed that marginally higher number of dystrophin-positive fibres was found in muscles treated withPNA30 AOs compared to PNA25 AOs, but the overall level was extremely low..3. One observation from above systemic studies is that mice treated with either PNA30 or PNA25 AOs displayed swollen tails, which prevented further investigation on them at higher doses. Subsequent examination revealed that PNA30 and PNA25 AOs solution was highly acidic and the acidity increased with the PNA length extended. Notably, the acidity of PNAAOs solution influences the activity of PNA AOs, with p H=2-3 being the optimal p H for the highest activity of PNAAOs.4. Given the low level of exon-skipping and dystrophin restoration observed with PNA25 AOs at the dose of 25 mg/kg, we increased the dose of PNA25 AOs to the highest dose we can test in mdx mice. The results showed that an increase in the number of dystrophin-positive fibres was detected in mdx mice treated with PNA25 AOs compared to PNA20 AOs under the identical dosing regimen of 50mg/kg for 3 weekly intravenous injections. However the level of dystrophin protein was lower than the therapeutic amount of dystrophin protein required for functional improvement. Therefore we increased the dose of PNA20 AOs from 50mg/kg to 100 mg/kg and compared to PNA18 AOs under the identical dosing regimen. The results showed that the number of dystrophin-positive fibers,exon-skipping efficiency and the level of dystrophin protein expression were higher in samples treated with PNA20 AOs than counterparts treated with PNA18 AOs.5. Subsequently, comprehensive investigation of PNA20 AOs in mdx mice revealed that the cumulative effect was achieved with repeated administrations of PNA20 AOs in mdx mice at the dose of 100 mg/kg for 5 weeks at weekly interval.Significant improvement in the grip strength was detected in mdx mice treated with repeated injections of PNA20 AOs, with correct re-localization of DAPC and improvement in biochemical parameters including serum CK, AST and ALT. No detectable drug-associated toxicity was observed in mdx mice with repeated administrations of PNA20 AOs, suggesting that PNA20 is the optimal combination of activity, solubility and safety.Conclusion:1. Optimization studies on different lengths of PNA AOs revealed that PNA AOs function in a length-dependent manner with PNA30 being significantly more potent than PNA25 AOs in inducing exon-skipping and dystrophin restoration.2. Systemic studies on PNA30 and PNA25 AOs in mdx mice indicated that acidity is an important parameter affecting the solubility and activity as well as safety of PNA AOs, with p H values ranging from 2 to 3 being the optimal p H for the activity of PNA AOs. The acidity observed in the PNA AO solution attributes to the content of Trifluoroacetic acid(TFA) used for the purification of PNA AOs.However the strong acidity in the long PNA AO solution(PNA30 and PNA25)preclude further investigation on PNA30 and PNA25 AOs in mdx mice at higher doses.3. Further systemic investigation on PNA20 AOs demonstrated that PNA AOs indeed can induce effective exon-skipping and dystrophin restoration in mdx mice with repeated administrations at the dose of 100 mg/kg intravenously.Therapeutics level of dystrophin protein can be achieved in mdx mice with PNA AOs at higher doses, resulting in functional and phenotypic rescue without any detectable toxicity, indicating PNA can be a viable chemistry for DMD exon-skipping therapeutics.