| Objective: Duchenne muscular dystrophy(DMD) is a X-linked fatal neuromuscular disorder with no racial difference worldwide. The disease is caused by mutations in the dystrophin gene, which results the absence of functional dystrophin protein. Currently there is no treatment available in the clinic. Antisense oligonucleotide(AO) mediated exon-skipping therapeutics shows promise and also one of the most extensively studied approaches for treating DMD. However, phase III clinical trial failed to meet the primary endpoints and it is likely attributed to the insufficient systemic delivery as demonstrated in pre-clinical studies. This highlights the urgency of developing safe and effective delivery formulations, compatible with the AO development. Previously, we identified a series of hexose-based novel formulations. Among them, GF showed the potential in enhancing the potency of PMO in body-wide peripheral muscles in mdx mice. However, the effect of GF in cardiac muscles remained limited. Heart failure accounts for the mortality of most DMD patients with the improvement in palliative care. In our previous studies, we developed a series of cell-penetrating peptide, which can significantly enhance systemic correction of dystrophin expression in body-wide muscles and cardiac tissues in adult mdx mice when conjugated with PMO. In this study, we aim to test whether GF can enhance the delivery of these PPMO drugs in mdx mice, particularly for cardiac and smooth muscles. Three representative PPMO drugs including R-PMO,Pip5e-PMO and B-MSP-PMO were chosen and tested with GF in mdx mice systemically.Methods:1. Delivery routes and dosing regimens: Single intravenous injection was used for the animal experiments throughout. 6-8-week old mdx mice were used for the study(3 mice per group). The dosing regimen for different AOs is as follows: R-PMO at12.5mg/kg dose and tissues were harvested 2 weeks after injection; 25mg/kg R-PMO and tissues were harvested 3 weeks after injection; 15mg/kg Pip5e-PMO and tissues were harvested 1 weeks after injection;, 20mg/kg Pip5e-PMO and tissues were harvested 2 days after injection; 20mg/kg B-MSP-PMO and tissues were harvested 2weeks after injection; lissamine-labeled Pip5e-PMO was used for tissue distribution study. For GF induction group, GF was intravenously administered into mdx mice for 4 weeks followed by co-injection of B-MSP-PMO with GF one week later.2. Sample collection and storage: blood was collected from orbital vein Body-wide tissues were harvested at desired time-points and snap-frozen in liquid nitrogen. Liver and kidneys were fixed in Bouin's solution.3. Tissue distribution study of Pip5e-PMO: Fluorescence intensity in body-wide tissues was measure with IVIS 2 days after intravenous injection of Pip5e-PMO at20mg/kg.4. Immunohistochemistry(IHC) assay: IHC was used to measure the distribution and expression dystrophin-positive fibers in body-wide muscles.5. RT-PCR: RT-PCR was used to measure the level of exon23 skipping at RNA level.6. Measurement of dystrophin protein expression: Western blot was used to measure dystrophin expression in body-wide muscles.7. Muscle functional assay: Functional rescue in treated mdx mice was assayed with immunostaining of dystrophin-associated protein complex(DAPC), quantification of centrally nucleated myofibers in quadriceps and gastrocnemius, evaluation of levels of CK-MB, CK in serum and,staining of inflammatory cells in diaphragms and grip strength test.8. Toxicity profiles: Biochemical parameters including glucose, aspartate aminotransferase(AST) and Creatinine(CREA) were examined in serum and Routine H&E staining for liver and kidney were performed to examine the potential B-MSP-PMO related toxicity.Results:1. Tissue distribution for Pip5e-PMO showed that GF did not promote the uptake of Pip5e-PMO in body-wide muscles as demonstrated by IVIS fluorescence intensity.2. IHC results in skeletal muscles demonstrated that: 1) administration of R-PMO at12.5 mg/kg dose achieved near normal level of dystrophin expression and there was no difference between GF and saline groups. And similar to 12.5 mg/kg, near normal level of dystrophin expression was observed in R-PMO at 25mg/kg dose and there was no difference between saline and GF groups. 2) both 15mg/kg and 20mg/kg doses of Pip5e-PMO induced a limited number of dystrophin-positive fibers in skeletal muscles and there was no significant difference between these two groups.3. IHC results in smooth muscles demonstrated that: 1) R-PMO induced few dystrophin-positive fibers in intestines from mdx mice treated with both saline and GF; although a lightly higher number of dystrophin-positive fibers was detected in lungs from mdx mice treated with 25mg/kg R-PMO and there was no difference between saline and GF. Strikingly, a higher number of dystrophin-positive fibers were achieved in trachea treated with 25mg/kg R-PMO in GF compared with the saline group. 2) there was no expression of dystrophin-positive fibers in smooth muscles examined from mdx mice treated with Pip5e-PMO at any dosing regimen. 3) a substantial number of dystrophin-positive fibers was observed in intestines, lungs and trachea from mdx mice treated with 20mg/kg B-MSP-PMO in saline, GF or GF induction groups, but there was no difference between 3 treatment groups in intestine or lung. There was significantly increase in the number of dystrophin-positive fibers in trachea treated with B-MSP-PMO in GF or GF induction compared with the saline.4. IHC results in cardiac muscles demonstrated that: 1) the number of dystrophinpositive fibers in cardiac muscles was increased at higher dose of R-PMO, though there was no difference between GF and saline. 2) very few dystrophin-positive fibers were found in cardiac muscle treated with both 15mg/kg and 20mg/kg Pip5e-PMO and there was no difference between GF and saline. 3) more dystrophin-positive fibers were detected in cardiac muscles treated with B-MSP-PMO in GF or GF induction compared with the saline group.5. RT-PCR results in skeletal muscles demonstrated that: 1) complete exon23 skipping was detected in skeletal muscles treated with both R-PMO in GF and saline..2) levels of exon skipping were about 70% or 90% in skeletal muscles treated with15mg/kg or 20mg/kg Pip5e-PMO, respectively, and there was no difference between the saline and GF groups. 3) 100% exon skipping was induced in body-wide skeletal muscles treated with 20mg/kg B-MSP-PMO and there was no difference between GF and saline.6. RT-PCR results in smooth muscles demonstrated that: 1) higher level of exon skipping was detected in intestines and lungs treated with higher dose of R-PMO compared with lower dose, whereas no difference was found between GF and saline.Strikingly, higher levels of exon skipping were trachea treated with R-PMO in GF compared with the saline group at the dose of 25mg/kg. 2) low levels of exon skipping were detected in smooth muscles treated with Pip5e-PMO at both 15mg/kg and 20 mg/kg doses and there was no difference between the two groups. 3) higher levels of exon skipping were detected in intestines and trachea treated with 20mg/kg B-MSP-PMO compared with lungs and there was no difference between GF and GF induction in intestines and lungs compared with saline. Importantly, higher level of exon skipping was found in trachea treated with B-MSP-PMO in GF compared with the saline group.7. RT-PCR results in cardiac muscles demonstrated that: 1) there is no difference in levels of exon skipping between R-PMO in GF and saline though higher level of exon skipping was detected in cardiac muscles treated wit higher dose of R-PMO. 2) there is no difference observed in the level of exon skipping in cardiac muscles treated with Pip5e-PMO in GF or saline under both dosing regimen. 3) higher levels of exon skipping was induced in cardiac muscles treated by 20mg/kg B-MSP-PMO in GF or GF induction groups compared with the saline group.8. Western blot results in skeletal muscles demonstrated that: 1) on average, about70% of normal level of dystrophin protein was restored in skeletal muscles treated with 12.5mg/kg R-PMO and there was no difference between GF and saline groups.In addition, approximately 100% dystrophin expression was detected in skeletal muscles treated with 25mg/kg R-PMO in GF or saline. 2) about 50% of normal level of dystrophin expression was observed in skeletal muscles treated with 15mg/kg Pip5e-PMO in GF or saline, while approximately 10% was detected in skeletal muscles treated with 20mg/kg Pip5e-PMO in GF or saline. 3) about 80% dystrophin expression in skeletal muscles treated with 20mg/kg B-MSP-PMO in saline, GF, or GF induction groups..9. Western blot results for R-PMO and B-MSP-PMO in smooth muscles: 1) Less than 10% dystrophin expression was detected in intestines treated with 12.5mg/kg R-PMO in GF and saline, though more than 20% detected in 25mg/kg R-PMO in GF and saline. About 20% dystrophin expression was detected in lungs treated with25mg/kg R-PMO grouping GF and saline. Importantly, more than 50% dystrophin expression was observed in trachea treated with 25 mg/kg R-PMO in GF; in contrast,there was only 30% dystrophin expression in the saline group. 2) About 60% or 20%dystrophin expression were detected in intestines or lungs treated with 20mg/kg B-MSP-PMO, respectively. And there was no difference between 3 treatment groups.Significantly higher levels of dystrophin expression were detected in trachea treated with B-MSP-PMO in GF(60%) and GF induction(80%) when compared with saline(40%).10. Western blot results in cardiac muscles: 1) About 20% dystrophin expression was observed in cardiac muscles treated with 12.5mg/kg R-PMO, whereas approximately50% in cardiac muscles treated with 25mg/kg R-PMO. There was no difference between saline and GF at both doses. 2) About 10% or 5% dystrophin expression were restored in cardiac muscles treated with 15mg/kg or 20mg/kg Pip5e-PMO,respectively. But there was no difference between GF and saline groups. 3)Significantly higher levels of dystrophin expression were detected in cardiac muscles treated with B-MSP-PMO in GF(80%) or GF induction(80%) compared with the saline group(60%).11. Muscle functional assay for B-MSP-PMO: 1) Correct re-localization of DAPC proteins was detected in cardiac and skeletal muscles treated with B-MSP-PMO in GF and saline. 2) Biochemical measurement of CK-MB and CK in serum indicated that significant decreases in levels of CK-MB and CK in serum from mdx mice treated with B-MSP-PMO compared with untreated mdx controls, suggesting that B-MSP-PMO treatment improved myocardial cell membrane integrity.12. Toxicity analysis: Levels of AST, ALT, ALP, UA and CREA significantly decreased in serum from mdx mice treated with B-MSP-PMO compared with untreated mdx controls, but no difference was detected between GF and saline.Measurement of glucose in serum also indicated no change in the level of glucose between B-MSP-PMO in GF and saline. The H&E staining revealed no pathological change in liver and kidney morphology, indicating no obvious toxicity in liver and kidney.Conclusions:1. Systemic evaluation of GF with 3 different PPMOs in mdx mice demonstrated that GF functions differently in different contexts.2. With the saturation doses of R-PMO, the enhancement was not evident in skeletal muscles, whereas the effect was significantly improved in trachea, compared with saline.3. GF significantly improved the activity of B-MSP-PMO in skeletal muscles and trachea, particularly for cardiac muscles, compared with saline.4. Repeated administration of GF is beneficial and safe; therefore GF can be used as a delivery formulation for DMD therapeutics. |
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