Triazine-cored polymeric vectors for antisense oligonucleotide delivery in vitro and in vivo.

BACKGROUND: The polymer-based drug/gene delivery is promising for the treatment of inherent or acquire disease, because of the polymer's structural flexibility, larger capacity for therapeutic agent, low host immunogenicity and less cost. Antisense therapy is an approach to fighting genetic disorders or infections using antisense oligonucleotides (AOs). Unfortunately, the naked AOs showed low therapeutic efficacy in vivo and in clinical trial due to their poor cellular uptake and fast clearance in bloodstream. In this study, a series of triazine-cored amphiphilic polymers (TAPs) were investigated for their potential to enhance delivery of AOs, 2'-O-methyl phosphorothioate RNA (2'-OMePS) and phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. RESULTS: TAPs significantly enhanced AO-induced exon-skipping in a GFP reporter-based myoblast and myotube culture system, and observed cytotoxicity of the TAPs were lower than Endoporter, Lipofectamine-2000 or PEI 25K. Application of optimized formulations of TAPs with AO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in dystrophic mdx mice. The best ones for PMO and 2'-OMePS delivery have reached to 11-, 15-fold compared with the AO only in mdx mice, respectively. CONCLUSION: The study of triazine-cored amphiphilic polymers for AO delivery in vitro and in mdx mice indicated that the carrier's performances are related to the molecular size, compositions and hydrophilic-lipophilic balance (HLB) of the polymers, as well as the AO's structure. Improved exon-skipping efficiency of AOs observed in vitro and in mdx mice accompanied with low cytotoxicity demonstrated TAP polymers are potentials as safe and effective delivery carrier for gene/drug delivery.

Aminoglycoside Enhances the Delivery of Antisense Morpholino Oligonucleotides In Vitro and in mdx Mice.

Antisense oligonucleotide (AO) therapy has been the specific treatment for Duchenne muscular dystrophy, with ongoing clinical trials. However, therapeutic applications of AOs remain limited, particularly because of the lack of efficient cellular delivery methods imperative for achieving efficacy. In this study, we investigated a few aminoglycosides (AGs) for their potential to improve the delivery of antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. AGs had lower cytotoxicity compared with Endoporter, the currently most effective delivery reagent for PMO in vitro, and improved efficiency in PMO delivery 9- to 15-fold over PMO alone. Significant enhancement in systemic PMO-targeted dystrophin exon 23 skipping was observed in mdx mice, up to a 6-fold increase with AG3 (kanamycin) and AG7 (sisomicin) compared with PMO only. No muscle damage could be detected clearly with the test dosages. These results establish AGs as PMO delivery-enhancing agents for treating muscular dystrophy or other diseases.

Saponins enhance exon skipping of 2'-O-methyl phosphorothioate oligonucleotide in vitro and in vivo.

BACKGROUND: Antisense oligonucleotide (ASO)-mediated exon skipping has been feasible and promising approach for treating Duchenne muscular dystrophy (DMD) in preclinical and clinical trials, but its therapeutic applications remain challenges due to inefficient delivery. METHODS: We investigated a few Saponins for their potential to improve delivery performance of an antisense 2'-Omethyl phosphorothioate RNA (2'-OMePS) in muscle cells and in dystrophic mdx mice. This study was carried out by evaluating these Saponins' toxicity, cellular uptake, transduction efficiency in vitro, and local delivery in vivo for 2'-OMePS, as well as affinity study between Saponin and 2'-OMePS. RESULTS: The results showed that these Saponins, especially Digitonin and Tomatine, enhance the delivery of 2'-OMePS with comparable efficiency to Lipofectamine 2k (LF-2k) -mediated delivery in vitro. Significant performance was further observed in mdx mice, up to 10-fold with the Digitonin as compared to 2'-OMePS alone. Cytotoxicity of the Digitonin and Glycyrrhizin was much lower than LF-2k in vitro and not clearly detected in vivo under the tested concentrations. CONCLUSION: This study potentiates Saponins as delivery vehicle for 2'-OMePS in vivo for treating DMD or other diseases.

Saponins as Natural Adjuvant for Antisense Morpholino Oligonucleotides Delivery In Vitro and in mdx Mice.

Antisense oligonucleotide (AON) therapy for Duchenne muscular dystrophy has drawn great attention in preclinical and clinical trials, but its therapeutic applications are still limited due to inefficient delivery. In this study, we investigated a few saponins for their potential to improve delivery performance of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that these saponins, especially digitonin and tomatine, improve the delivery efficiency of PMO comparable to Endo-Porter-mediated PMO delivery in vitro. The significant enhancement of PMO targeting to dystrophin exon 23 delivery was further observed in mdx mice up to 7-fold with the digitonin as compared to PMO alone. Cytotoxicity of the digitonin and glycyrrhizin was lower than Endo-Porter in vitro and not clearly detected in vivo under the tested concentrations. These results demonstrate that optimization of saponins in molecular size and composition are key factors to achieve enhanced PMO exon-skipping efficiency. The higher efficiency and lower toxicity endow saponins as gene/AON delivery enhancing agents for treating muscular dystrophy or other diseases.

Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo- mouse model of FKRP-deficient muscular dystrophy.

BACKGROUND: Fukutin-related protein (FKRP) mutations are the most common cause of dystroglycanopathies known to cause both limb girdle and congenital muscular dystrophy. The P448Lneo- mouse model has a knock-in mutation in the FKRP gene and develops skeletal, respiratory, and cardiac muscle disease. METHODS: We studied the natural history of the P448Lneo- mouse model over 9 months and the effects of twice weekly treadmill running. Forelimb and hindlimb grip strength (Columbus Instruments) and overall activity (Omnitech Electronics) assessed skeletal muscle function. Echocardiography was performed using VisualSonics Vevo 770 (FujiFilm VisualSonics). Plethysmography was performed using whole body system (ADInstruments). Histological evaluations included quantification of inflammation, fibrosis, central nucleation, and fiber size variation. RESULTS: P448Lneo- mice had significantly increased normalized tissue weights compared to controls at 9 months of age for the heart, gastrocnemius, soleus, tibialis anterior, quadriceps, and triceps. There were no significant differences seen in forelimb or hindlimb grip strength or activity monitoring in P448Lneo- mice with or without exercise compared to controls. Skeletal muscles demonstrated increased inflammation, fibrosis, central nucleation, and variation in fiber size compared to controls (p < 0.05) and worsened with exercise. Plethysmography showed significant differences in respiratory rates and decreased tidal and minute volumes in P448Lneo- mice (p < 0.01). There was increased fibrosis in the diaphragm compared to controls (p < 0.01). Echocardiography demonstrated decreased systolic function in 9-month-old mutant mice (p < 0.01). There was increased myocardial wall thickness and mass (p < 0.001) with increased fibrosis in 9-month-old P448Lneo- mice compared to controls (p < 0.05). mRNA expression for natriuretic peptide type A (Nppa) was significantly increased in P448Lneo- mice compared to controls at 6 months (p < 0.05) and for natriuretic peptide type B (Nppb) at 6 and 9 months of age (p < 0.05). CONCLUSIONS: FKRP-deficient P448Lneo- mice demonstrate significant deficits in cardiac and respiratory functions compared to control mice, and this is associated with increased inflammation and fibrosis. This study provides new functional outcome measures for preclinical trials of FKRP-related muscular dystrophies.

Distinct expression of functionally glycosylated alpha-dystroglycan in muscle and non-muscle tissues of FKRP mutant mice.

The glycosylation of alpha-dystroglycan (α-DG) is crucial in maintaining muscle cell membrane integrity. Dystroglycanopathies are identified by the loss of this glycosylation leading to a breakdown of muscle cell membrane integrity and eventual degeneration. However, a small portion of fibers expressing functionally glycosylated α-DG (F-α-DG) (revertant fibers, RF) have been identified. These fibers are generally small in size, centrally nucleated and linked to regenerating fibers. Examination of different muscles have shown various levels of RFs but it is unclear the extent of which they are present. Here we do a body-wide examination of muscles from the FKRP-P448L mutant mouse for the prevalence of RFs. We have identified great variation in the distribution of RF in different muscles and tissues. Triceps shows a large increase in RFs and together with centrally nucleated fibers whereas the pectoralis shows a reduction in revertant but increase in centrally nucleated fibers from 6 weeks to 6 months of age. We have also identified that the sciatic nerve with near normal levels of F-α-DG in the P448Lneo- mouse with reduced levels in the P448Lneo+ and absent in LARGEmyd. The salivary gland of LARGEmyd mice expresses high levels of F-α-DG. Interestingly the same glands in the P448Lneo-and to a lesser degree in P448Lneo+ also maintain considerable amount of F-α-DG, indicating the non-proliferating epithelial cells have a molecular setting permitting glycosylation.

Tween 85-Modified Low Molecular Weight PEI Enhances Exon-Skipping of Antisense Morpholino Oligomer In Vitro and in mdx Mice.

We investigated a series of Tween 85 modified low molecular weight polyethylenimine (LPEI, 0.8k/1.2k/2.0k)-copolymers (Zs) through simple formulation and covalent conjugation with phosphorodiamidate morpholino oligomer (PMO) for their potential to enhance delivery in vitro and in dystrophic mdx mice. Z polymers significantly enhanced PMO-induced exon-skipping in a GFP reporter-based cell culture system. Application of optimized formulations of Zs with PMO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in mdx mice. Consistent with our observations in vitro, optimization of molecular size and hydropholic-lipopholic balance (HLB) of polymers are important factors to achieve enhanced PMO delivery in vivo. The best formulation of Zs enhanced PMO delivery with 20- and 6-fold over PMO alone in vitro and in vivo, respectively. Further, chemical conjugation of the polymer and PMO exhibits greater benefit than polymer/PMO simple formulation in PMO delivery efficiency. Observed cytotoxicity of the Zs was lower than Endo-porter and PEI 25k in vitro, and no tissue toxicity was clearly detected with the Zs at the dosage tested. These results indicate the potential of the Zs as effective and safe PMO delivery carriers for treating diseases such as muscular dystrophy.

Autologous intramuscular transplantation of engineered satellite cells induces exosome-mediated systemic expression of Fukutin-related protein and rescues disease phenotype in a murine model of limb-girdle muscular dystrophy type 2I.

α-Dystroglycanopathies are a group of muscular dystrophies characterized by α-DG hypoglycosylation and reduced extracellular ligand-binding affinity. Among other genes involved in the α-DG glycosylation process, fukutin related protein (FKRP) gene mutations generate a wide range of pathologies from mild limb girdle muscular dystrophy 2I (LGMD2I), severe congenital muscular dystrophy 1C (MDC1C), to Walker-Warburg Syndrome and Muscle-Eye-Brain disease. FKRP gene encodes for a glycosyltransferase that in vivo transfers a ribitol phosphate group from a CDP -ribitol present in muscles to α-DG, while in vitro it can be secreted as monomer of 60kDa. Consistently, new evidences reported glycosyltransferases in the blood, freely circulating or wrapped within vesicles. Although the physiological function of blood stream glycosyltransferases remains unclear, they are likely released from blood borne or distant cells. Thus, we hypothesized that freely or wrapped FKRP might circulate as an extracellular glycosyltransferase, able to exert a "glycan remodelling" process, even at distal compartments. Interestingly, we firstly demonstrated a successful transduction of MDC1C blood-derived CD133+ cells and FKRP L276IKI mouse derived satellite cells by a lentiviral vector expressing the wild-type of human FKRP gene. Moreover, we showed that LV-FKRP cells were driven to release exosomes carrying FKRP. Similarly, we observed the presence of FKRP positive exosomes in the plasma of FKRP L276IKI mice intramuscularly injected with engineered satellite cells. The distribution of FKRP protein boosted by exosomes determined its restoration within muscle tissues, an overall recovery of α-DG glycosylation and improved muscle strength, suggesting a systemic supply of FKRP protein acting as glycosyltransferase.

Polyquaternium-mediated delivery of morpholino oligonucleotides for exon-skipping in vitro and in mdx mice.

Antisense oligonucleotide therapy for Duchenne muscular dystrophy has shown great potential in preclinical and clinical trials, but its therapeutic applications are still limited due to inefficient delivery. In this study, we investigated a few polyquaterniums (PQs) with different size and composition for their potential to improve delivery performance of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that LuviquatTM series, especially PQ-1 and PQ-3, promoted the exon-skipping efficiency comparable to Endoporter-mediated PMO delivery in vitro. Significant enhancement in skipping dystrophin exon 23 has also been achieved with PQ-3 up to seven-fold when compared to PMO alone in mdx mice. Cytotoxicity of the PQs was lower than Endoporter and PEI 25 K in vitro and muscle damage not clearly detected in vivo under the tested concentrations. These results together demonstrate that the optimization of PQ in molecular size, composition and distribution of positive charges is the key factor to achieve enhanced PMO exon-skipping efficiency. The higher efficiency and lower toxicity endow polyquaternium series as AO delivery enhancing agents for treating muscular dystrophy and other diseases.

Evaluation of Amphiphilic Peptide Modified Antisense Morpholino Oligonucleotides In Vitro and in Dystrophic mdx Mice.

A series of amphiphilic peptides modified PMO (Pt-PMO) were prepared, and their antisense effect and toxicity were evaluated both in vitro and in mdx mice. The results showed that the exon-skipping performance of Pt-PMO are relative to the structure of the conjugated peptide: the Pt3/Pt4 composed of six/seven arginines and one myristoylation modified PMO showed more efficacy and with less toxicity as compared to others, confirming that appropriate hydrophilic-lipophilic balance (HLB) and cationic sequence numbers play a crucial role in improving cell uptake and corresponding exon-skipping efficiency. This was observed particularly in enhanced delivery efficiency of PMO comparable to B-PMO in vitro, while 6-fold improved exon-skipping was achieved against naked PMO in vivo. The multi-PMO modified Pt8-PMO also showed improved exon-skipping both in vitro and in vivo, though there is lower efficiency in systemic delivery as compared to Pt4-PMO. These data suggest that with optimization of peptide in component, charge density has clear potential for exploration towards achieving higher efficiency of antisense oligonucleotide systemic delivery, and thus is more applicable for clinical application.

A combinatorial library of triazine-cored polymeric vectors for pDNA delivery in vitro and in vivo.

A set of triazine-cored cationic amphiphilic polymers (TAPs) composed of low molecular weight (Mw) polyethylenimine (LPEI, B) and amphiphilic Jeffamine (A) were prepared with controllable composition and molecular size, and further characterized for plasmid DNA (pDNA) delivery both in vitro and in vivo. These new polymers condensed pDNA efficiently at a polymer/pDNA weight ratio of 5 with particle sizes below 200 nm. The introduction of Jeffamine in the polymers significantly improved the cellular uptake of pDNA, but without increasing its toxicity compared with the parent LPEI. The best formulation resulted in 6- and 29-fold transfection efficiencies of PEI 25k in vitro and in vivo in mdx mice, respectively. Higher transfection efficiency was achieved with more lipophilic A1/A3-based polymers in vitro, with 1A11B3 and 1A12B3 showing the greatest delivery performance. However, the lipophilicity of the TAPs is less critical in vivo as the less lipophilic A2/A4 constructed TAPs also performed similarly well as the more lipophilic A1/A3 constructed ones. In addition, a synergistic effect of LPEI and Jeffamine via chemical conjugation for the delivery of pDNA was revealed in transfection efficiency. These results indicate that the appropriate positive surface and particle size of polymer/pDNA complex and the composition and hydrophilic-lipophilic balance (HLB) of polymers are crucial for effective delivery, although intricate matching exists between A and B in the TAP composition. Triazine-cored cationic amphiphilic polymers are safe and potentially effective carriers for gene/drug delivery.

Progressive Dystrophic Pathology in Diaphragm and Impairment of Cardiac Function in FKRP P448L Mutant Mice.

Mutations in the gene for fukutin-related protein represent a subset of muscular dystrophies known as dystroglycanopathies characterized by loss of functionally-glycosylated-alpha-dystroglycan and a wide range of dystrophic phenotypes. Mice generated by our lab containing the P448L mutation in the fukutin-related protein gene demonstrate the dystrophic phenotype similar to that of LGMD2I. Here we examined the morphology of the heart and diaphragm, focusing on pathology of diaphragm and cardiac function of the mutant mice for up to 12 months. Both diaphragm and heart lack clear expression of functionally-glycosylated-alpha-dystroglycan throughout the observed period. The diaphragm undergoes progressive deterioration in histology with increasing amount of centranucleation and inflammation. Large areas of mononuclear cell infiltration and fibrosis of up to 60% of tissue area were detected as early as 6 months of age. Despite a less severe morphology with only patches of mononuclear cell infiltration and fibrosis of ~5% by 12 months of age in the heart, cardiac function is clearly affected. High frequency ultrasound reveals a smaller heart size up to 10 months of age. There are significant increases in myocardial thickness and decrease in cardiac output through 12 months. Dysfunction in the heart represents a key marker for evaluating experimental therapies aimed at cardiac muscle.

Poly(ester amine) Composed of Polyethylenimine and Pluronic Enhance Delivery of Antisense Oligonucleotides In Vitro and in Dystrophic mdx Mice.

A series of poly(esteramine)s (PEAs) constructed from low molecular weight polyethyleneimine (LPEI) and Pluronic were evaluated for the delivery of antisense oligonuclotides (AOs), 2'-O-methyl phosphorothioate RNA (2'-OMePS) and phosphorodiamidate morpholino oligomer (PMO) in cell culture and dystrophic mdx mice. Improved exon-skipping efficiency of both 2'-OMePS and PMO was observed in the C2C12E50 cell line with all PEA polymers compared with PEI 25k or LF-2k. The degree of efficiency was found in the order of PEA 01, PEA 04 > PEA 05 > others. The in vivo study in mdx mice demonstrated enhanced exon-skipping of 2'-OMePS with the order of PEA 06 > PEA 04, PEA 07 > PEA 03 > PEA 01 > others, and much higher than PEI 25k formulated 2'-OMePS. Exon-skipping efficiency of PMO in formulation with the PEAs were significantly enhanced in the order of PEA 02 > PEA 10 > PEA 01, PEA 03 > PEA 05, PEA 07, PEA 08 > others, with PEA 02 reaching fourfold of Endo-porter formulated PMO. PEAs improve PMO delivery more effectively than 2'-OMePS delivery in vivo, and the systemic delivery evaluation further highlight the efficiency of PEA for PMO delivery in all skeletal muscle. The results suggest that the flexibility of PEA polymers could be explored for delivery of different AO chemistries, especially for antisense therapy.

Poly(ester amine) constructed from polyethylenimine and pluronic for gene delivery in vitro and in vivo.

A series of poly (ester amines) (PEAs) constructed from low molecular weight polyethyleneimine (LPEI, Mw: 0.8k, 1.2k Da) and Pluronic (different molecular weight (Mw) and hydrophilic-lipophilic-balance (HLB)) components were synthesized, and evaluated in vitro and in vivo as gene delivery carriers. Most PEA polymers were able to bind and condense plasmid DNA effectively into particles of approximately 150 nm in solution at the polymer/DNA ratio of 5 and above. Transfection efficiency of the PEA polymers depends on particle size of the polymer/DNA complex, molecular weight and HLB of the Pluronics and the size of PEI within PEA composition, as well as the cell type. Significant improvement in gene delivery efficacy was achieved with PEA01/04/05 composed of Pluronic size (Mw: 3000-5000 Da), and HLB (12-18) in CHO, C2C12 and HSkM cell lines; and the effective transfection was reflected with PEA 01/04/07 composed of Pluronics with size (2000-5000 Da) and HLB (12-23) in mdx mice. The best formulation for pDNA delivery was obtained with PEA 01 producing transgene expression efficiency 5, 19-folds of that of PEI 25k in vitro and in vivo, respectively. These results potent some of these PEA polymers as attractive vehicles for gene or oligonucleotide delivery.

Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice.

In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases.

Tween 85 grafted PEIs enhanced delivery of antisense 2'-O-methyl phosphorothioate oligonucleotides in vitro and in dystrophic mdx mice.

A series of cationic amphiphlic copolymers (Z series) constructed from Tween 85 and low molecular weight (Mw) polyethyleneimine (LPEI) have been evaluated for the delivery of antisense 2'-O-methyl phosphorothioate RNA (2'-OMePS) in both cell culture and dystrophic mdx mice. All Z copolymers improved the 2'-OMePS-induced dystrophin expression both in vitro and in vivo compared with PEI 25k formulated or 2'-OMePS alone. The most effective polymers are in the order of Z9 > Z3 > Z7, Z1, Z2, Z6 > others by formulation at the dose of 20 µg mL-1 in myoblast cell culture. Significantly enhanced exon-skipping of 2'-OMePS with Z polymers in mdx mice was obtained in the order of Z7 > Z9, Z3 > Z8, Z6 > others. The highest efficiency of targeted exon-skipping with Z7 [T85-PEI 2k (1 : 1)] reached over 8 fold compared with 2'-OMePS alone in mdx mice. Further analyses of the structure and function indicate that the more hydrophobicity and lower PEI content of the polymer microstructure are, the greater are the delivery efficiency and exon-skipping. The unique hydrophobic interactions between the Z polymers and 2'-OMePS likely create more stable complexes in primarily hydrophilic environments both in vitro and in vivo. The overall results suggested that Tween 85 modified LPEIs provide a promising delivery approach for applications of 2'-OMePS oligonucleotides as therapeutic reagents.

Muscle and heart function restoration in a limb girdle muscular dystrophy 2I (LGMD2I) mouse model by systemic FKRP gene delivery.

Mutations in fukutin-related protein (FKRP) gene cause a wide spectrum of disease phenotypes including the mild limb-girdle muscular dystrophy 2I (LGMD2I), the severe Walker-Warburg syndrome, and muscle-eye-brain disease. FKRP deficiency results in α-dystroglycan (α-DG) hypoglycosylation in the muscle and heart, which is a biochemical hallmark of dystroglycanopathies. To study gene replacement therapy, we generated and characterized a new mouse model of LGMD2I harboring the human mutation leucine 276 to isoleucine (L276I) in the mouse alleles. The homozygous knock-in mice (L276I(KI)) mimic the classic late onset phenotype of LGMD2I in both skeletal and cardiac muscles. Systemic delivery of human FKRP gene by AAV9 vector in the L276I(KI) mice, at either neonatal age or at the age of 9 months, rendered body wide FKRP expression and restored glycosylation of α-DG in both skeletal and cardiac muscles. FKRP gene therapy ameliorated dystrophic pathology and cardiomyopathy such as muscle degeneration, fibrosis, and myofiber membrane leakage, resulting in restoration of muscle and heart contractile functions. Thus, these results demonstrated that the treatment based on FKRP gene replacement was effective.

A nitrate ester of sedative alkyl alcohol improves muscle function and structure in a murine model of Duchenne muscular dystrophy.

Nitric oxide (NO) has major physiological and cellular effects on muscle growth, repair, and function. In most muscle biopsies from humans with myopathies, sarcolemma-localized neuronal nitric oxide synthase (nNOS) is either reduced or not detected, particularly in dystrophin-deficient Duchenne muscular dystrophy (DMD). Abnormal NO signaling at the sarcolemmal level is integrally involved in the pathogenesis and accounts, at least in part, for the muscle weakness of DMD. Dystrophic muscle fibers exhibit an increased susceptibility to contraction-induced membrane damage. Muscle relaxants function to prevent muscle wasting by decreasing nerve impulses and reducing calcium influx that regulates tensing or tightening of muscle fibers. We have recently developed a new class of nitric esters that combines the pharmacological functions of NO and muscle relaxation. Here, we report the synthesis and properties of the nitric ester (MMPN) of 2-methyl-2-n-propyl-1,3-propanediol (MPP) and its effect in mdx dystrophic mice, a murine model of DMD. MMPN produced significant improvements in biochemical, pathological, and functional phenotypes in the mouse model. The endurance of exercise was extended by 47% in time to exhaustion and 84% in running distance. Serum CK level was decreased by 30%. Additionally, MMPN decreased intracellular free calcium concentration without causing skeletal muscle weakness. No hepatic or renal toxicities were observed during the study. Our investigations unveil a potential new treatment for muscular diseases.

Polyethylenimine-modified pluronics (PCMs) improve morpholino oligomer delivery in cell culture and dystrophic mdx mice.

We investigated a series of small-sized polyethylenimine (PEI, 0.8/1.2 k)-conjugated pluronic copolymers (PCMs) for their potential to enhance delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in dystrophic mdx mice. PCM polymers containing pluronics of molecular weight (Mw) ranging 2-6 k, with hydrophilic-lipophilic balance (HLB) 7-23, significantly enhanced PMO-induced exon-skipping in a green fluorescent protein (GFP) reporter-based myoblast culture system. Application of optimized formulations of PCMs with PMO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in dystrophic mdx mice. Consistent with our observations in vitro, optimization of molecular size and the HLB of pluronics are important factors for PCMs to achieve enhanced PMO delivery in vivo. Observed cytotoxicity of the PCMs was lower than Endo-porter and PEI 25 k. Tissue toxicity of PCMs in muscle was not clearly detected with the concentrations used, indicating the potential of the PCMs as effective and safe PMO carriers for treating diseases such as muscular dystrophy.