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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., Graphical Abstract

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.

Mingxing Wang, Bo Wu, Sapana N Shah, Peijuan Lu, Qilong Lu McColl-Lockwood Laboratory for Muscular Dystrophy Research, Department of Neurology, Cannon Research Center, Carolinas Medical Center, Charlotte, NC 28203, USA 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. Keywords: antisense delivery, 2′-OMePS, exon skipping, saponin, muscular dystrophy

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., Graphical Abstract

Loss-of-function mutations in the Fukutin-related protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) and other forms of congenital muscular dystrophy-dystroglycanopathy that are associated with glycosylation defects in the α-dystroglycan (α-DG) protein. Systemic administration of a single dose of recombinant adeno-associated virus serotype 9 (AAV9) vector expressing human FKRP to a mouse model of LGMD2I at various stages of disease progression was evaluated. The results demonstrate rescue of functional glycosylation of α-DG and muscle function, along with improvements in muscle structure at all disease stages versus age-matched untreated cohorts. Nevertheless, mice treated in the latter stages of disease progression revealed a decrease in beneficial effects of the treatment. The results provide a proof of concept for future clinical trials in patients with FKRP-related muscular dystrophy and demonstrate that AAV-mediated gene therapy can potentially benefit patients at all stages of disease progression, but earlier intervention would be highly preferred.

Additional file 1. 1) Polymer’s structure and corresponding code; 2) Comparison of transduction and cytotoxicity between TAP (10 μg) and LF-2k (4 μg) mediated 2′-OMePSE50.

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.

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.

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.

Mingxing Wang, Bo Wu, Jason D Tucker, Peijuan Lu, Qilong Lu Department of Neurology, McColl-Lockwood Laboratory forMuscular Dystrophy Research, Cannon Research Center, Carolinas Medical Center, Charlotte, NC, USA Abstract: 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 polyethylenimine25,000 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. Keywords: cationic polyelectrolytes, antisense delivery, exon-skipping, PMO, muscular dystrophy

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.

The third most common form of limb-girdle muscular dystrophies is caused by mutations of the Fukutin-related protein ( FKRP ) gene, with no effective therapy available. Selective estrogen receptor modulators, tamoxifen and raloxifene, have been widely used for human conditions for their anti-inflammatory, antifibrosis, prevention of bone loss, and muscle building effects (essential features for muscular dystrophy therapies). We evaluated therapeutic values of tamoxifen and raloxifene in FKRPP448L mutant mouse with severe dystrophic phenotype. The mice were treated with the drugs for 1 year through daily gavage. We demonstrate that tamoxifen and raloxifene significantly ameliorated the disease progression. The improvement includes increase in grip force production, extended running time and distance in treadmill test, and enhancement in cardiac and respiratory functions. Significant reduction in muscle pathology includes diminished fibrosis and fiber degeneration. Tamoxifen and raloxifene also significantly mitigated bone loss. Tamoxifen, but not raloxifene, caused severe adverse effects on male reproductive organs. The results demonstrate that tamoxifen and raloxifene hold significant potential for treating FKRP -related muscular dystrophy and probably other muscular dystrophies. Sex-related differential effects of the drugs call for a careful consideration for the drug and dosage selection in male and female patient populations.

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.

Hyperbranched poly(ester amine)s (PEAs) based on tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) cross-linked low-molecular-weight polyethylenimine (Mw: 0.8k/1.2k/2.0k) have been evaluated for delivering antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in vivo in the dystrophic mdx mouse. The results show that the PEAs constructed with polyethylenimine (PEI) 2.0k (C series) improved PMO delivery more efficiently than those constructed with PEI 0.8k (A series) or 1.2k (B series) in a GFP reporter-based C2C12 mouse myoblast culture system. The highest efficiency of exon-skipping in vitro with the PMO oligonucleotide targeting human dystrophin exon 50 was obtained when the PEA C12 [TAEI-PEI 2.0k (1:2)] was used. Nearly all of the PEAs improved dystrophin expression in mdx mice by local injection with a 2–4-fold increase when compared with PMO alone. Improved transfection efficiency and lower toxicity indicate the potential of the biodegradable PEA polymers as safe and efficient PMO delivery vectors for in vivo applications.

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.

Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by mutations in the fukutin-related protein (FKRP) gene. Unlike its severe allelic forms, LGMD2I usually involves slower onset and milder course without defects in the central nervous system. The lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes led us to use RNA interference technology to knock down FKRP expression via postnatal gene delivery so as to circumvent embryonic lethality. Specifically, an adeno-associated viral vector was used to deliver short hairpin (shRNA) genes to healthy ICR mice. Adeno-associated viral vectors expressing a single shRNA or two different shRNAs were injected one time into the hind limb muscles. We showed that FKRP expression at 10 months postinjection was reduced by about 50% with a single shRNA and by 75% with the dual shRNA cassette. Dual-cassette injection also reduced a-dystroglycan glycosylation and its affinity to laminin by up to 70% and induced α-dystrophic pathology, including fibrosis and central nucleation, in more than 50% of the myofibers at 10 months after injection. These results suggest that the reduction of approximately or more than 75% of the normal level of FKRP expression induces chronic dystrophic phenotypes in skeletal muscles. Furthermore, the restoration of about 25% of the normal FKRP level could be sufficient for LGMD2I therapy to correct the genetic deficiency effectively and prevent dystrophic pathology.

Exon skipping has demonstrated great potential for treating Duchenne muscular dystrophy (DMD) and other diseases. We have developed a drug-screening system using C2C12 myoblasts expressing a reporter green fluorescent phosphate (GFP), with its reading frame disrupted by the insertion of a targeted dystrophin exon. A library of 2,000 compounds (Spectrum collection; Microsource Discovery System) was screened to identify drugs capable of skipping targeted dystrophin exons or enhancing the exon-skipping effect by specific antisense oligomers. The 6-thioguanine (6TG) was effective for inducing skipping of both human dystrophin exon 50 (hDysE50) and mouse dystrophin exon 23 (mDysE23) in the cell culture systems and increased exon skipping efficiency (more than threefolds) when used in combination with phosphorodiamidate morpholino oligomers (PMO) in both myoblasts and myotubes. Guanine and its analogues were unable to induce detectable skipping of exon 23 when used alone but enhanced PMO-induced exon skipping significantly (approximately two times) in the muscles of dystrophic mdx mouse in vivo. Our results demonstrate that small-molecule compounds could enhance specific exon skipping synergistically with antisense oligomers for experimental therapy to human diseases.

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.

Fukutin-related protein-muscular dystrophy is characterized by defects in glycosylation of α-dystroglycan with variable clinical phenotypes, most commonly as limb-girdle muscular dystrophy 2I. There is no effective therapy available. Glucocorticoid steroids have become the standard treatment for Duchenne and other muscular dystrophies with serious adverse effects, including excessive weight gain, immune suppression, and bone loss. Bisphosphonates have been used to treat Duchenne muscular dystrophy for prevention of osteoporosis. Herein, we evaluated prednisolone and alendronate for their therapeutic potential in the FKRPP448L- mutant mouse representing moderate limb-girdle muscular dystrophy 2I. Mice were treated with prednisolone, alendronate, and both in combination for up to 6 months. Prednisolone improved muscle pathology with significant reduction in muscle degeneration, but had no effect on serum creatine kinase levels and muscle strength. Alendronate treatment did not ameliorate muscle degeneration, but demonstrated a limited enhancement on muscle function test. Combined treatment of prednisolone and alendronate provided best improvement in muscle pathology with normalized fiber size distribution and significantly reduced serum creatine kinase levels, but had limited effect on muscle force generation. The use of alendronate significantly mitigated the bone loss. Prednisolone alone and in combination with alendronate enhance functionally glycosylated α-dystroglycan. These results, for the first time, demonstrate the efficacy and feasibility of this alliance treatment of the two drugs for fukutin-related protein-muscular dystrophy.

Branched poly(ester amine)s (PEAs) based on tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) cross-linked low molecular weight polyethylenimine (LPEI, Mw: 0.8k/1.2k/2.0k) polymers have been evaluated for their ability to deliver antisense 2′-O-methyl phosphorothioate RNA (2′-OMePS) through in vitro and in dystrophic mdx mice. Almost all PEAs improved exon-skipping of 2′-OMePS both in vitro and in vivo. Especially the PEAs composed of PEI 2.0k (C series) enhanced 2′-OMePS delivery more effectively than PEI 0.8k or 1.2k-based series using a GFP reporter-based C2C12 myoblast culture system and in mdx mice. The highest efficiency of targeted exon-skipping of 2′-OMePS was obtained by PEA C14 [TAEI-PEI 2.0k (1:4)] with up to 6 fold increase compared with 2′-OMePS only in mdx mice via intramuscular injection without increased muscle damage. Enhanced exon-skipping and lower toxicity highlight the potential of this series biodegradable PEAs as safe and efficient 2′-OMePS delivery vectors for treating diseases such as muscular dystrophy with splicing modulation.

A series of cationic amphiphlic copolymers constructed from Tween 85 and low molecular weight (Mw) polyethyleneimene (LPEI) were prepared in ambient conditions and characterized. The Mws of these copolymers ranged from around 5,000 to 25,000 Da and PEI content from 8.25% to 20.91%. The new copolymers condensed DNA efficiently with particles size below 200 nm at the weight ratio 5 of polymer/pDNA, and were stable in the presence of Serum or Heparin. The introduction of Tween 85 led to a significant increase in the cellular uptake of complexes with higher transfection efficiency in CHO, C2C12, and HSkM cell lines, but without increase in toxicity compared with the parent LPEI. The best formulation for pDNA delivery produced transgene expression efficiency 5, 20-fold of PEI 25k in vitro and in mdx mice in vivo, respectively. There is no obvious muscle damage with these new copolymers at the injection sites. These results indicated that the presence of more hydrophobic groups within the new polymers is associated with higher transfection efficiency (TE), and the higher PEI content and Mw of polymers show higher TE with relatively higher toxicity. The Tween 85 modified LPEI could be a potentially safe and effective polymeric carriers for gene/drug delivery.

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.

Exon skipping is capable of correcting frameshift and nonsense mutations in Duchenne muscular dystrophy. Phase 2 clinical trials in the United Kingdom and the Netherlands have reported induction of dystrophin expression in muscle of Duchenne muscular dystrophy patients by systemic administration of both phosphorodiamidate morpholino oligomers (PMO) and 2′-O-methyl phosphorothioate. Peptide-conjugated phosphorodiamidate morpholino offers significantly higher efficiency than phosphorodiamidate morpholino, with the ability to induce near-normal levels of dystrophin, and restores function in both skeletal and cardiac muscle. We examined 1-year systemic efficacy of peptide-conjugated phosphorodiamidate morpholino targeting exon 23 in dystrophic mdx mice. The LD50 of peptide-conjugated phosphorodiamidate morpholino was determined to be approximately 85 mg/kg. The half-life of dystrophin expression was approximately 2 months in skeletal muscle, but shorter in cardiac muscle. Biweekly injection of 6 mg/kg peptide-conjugated phosphorodiamidate morpholino produced >20% dystrophin expression in all skeletal muscles and ≤5% in cardiac muscle, with improvement in muscle function and pathology and reduction in levels of serum creatine kinase. Monthly injections of 30 mg/kg peptide-conjugated phosphorodiamidate morpholino restored dystrophin to >50% normal levels in skeletal muscle, and 15% in cardiac muscle. This was associated with greatly reduced serum creatine kinase levels, near-normal histology, and functional improvement of skeletal muscle. Our results demonstrate for the first time that regular 1-year administration of peptide-conjugated phosphorodiamidate morpholino can be safely applied to achieve significant therapeutic effects in an animal model.

Hyperbranched poly(ester amine)s (PEAs) were successfully synthesized by Michael addition reaction between tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) and low-molecular-weight polyethylenimine (LPEI, M(w) 0.8k, 1.2k, and 2.0k) and evaluated in vitro and in vivo as gene carriers. PEAs effectively condensed plasmid DNA with particle sizes below 200 nm and surface charges between 11.5 and 33.5 mV under tested doses [at the ratios 2-10:1 of polymer/pDNA(w/w)]. The PEAs showed significantly lower cytotoxicities when compared with PEI 25k in two different cell lines. The PEAs (C series) composed of PEI 2k showed higher transgene expression compared to PEAs of PEI 0.8k (A series) or 1.2k (B series). Highest gene transfection efficiency in CHO, C2C12 myoblast, and human skeletal muscle (HSK) cell lines was obtained with TAEI/PEI-2K (C12) at a ratio of 1:2. Both C12, C14(TAEI/PEI-2K at a ratio of 1:4) demonstrated 5-8-fold higher gene expression as compared with PEI 25k in mdx mice in vivo through intramuscular administration. No obvious muscle damage was observed with these new polymers. Higher transfection efficiency and lower toxicity indicate the potential of the biodegradable PEAs as safe and efficient transgene delivery vectors.

Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs). However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD/mdx mouse, a transgenic model carrying the full-length human dystrophin gene with mdx background, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD/mdx mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD/mdx mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement.

Antisense therapy has been successful to skip targeted dystrophin exon with correction of frameshift and nonsense mutations of Duchenne muscular dystrophy (DMD). Systemic production of truncated but functional dystrophin proteins has been achieved in animal models. Furthermore, phase I/II clinical trials in United Kingdom and the Netherlands have demonstrated dystrophin induction by local and systemic administrations of antisense oligomers. However, long-term efficacy and potential toxicity remain to be determined. The present study examined 1-year systemic effect of phosphorodiamidate morpholino oligomers (PMO) treatment targeting mutated dystrophin exon 23 in mdx mice. PMO induced dystrophin expression dose-dependently and significantly improved skeletal muscle pathology and function with reduced creatine kinase (CK) levels by a regimen of 60 mg/kg biweekly administration. This regimen induced

Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cell-penetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo . Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) results from dystrophin gene mutations, causing shift of the reading frame and preventing production of a functional protein. Most DMD mutations occur in the parts of the gene that are not critical for its function, therefore restoration of the reading frame by antisense oligonucleotide-mediated exon skipping is a viable approach. The efficacy of antisense therapy has now been proven in animal models and in clinical trials.Therapeutic effect of exon skipping largely depends on the efficiency of individual antisense oligoneotide, which has to be identified by screening. This study aims specifically to search for the most effective morpholino (PMO) oligomer to target the human dystrophin exon 51 and exon 53 for the correction of the relevant DMD mutations. We established the GFP reporter myoblast cell cultures and screened more than 40 PMOs targeting each of exon 51 and exon 53. We also examined the PMOs in normal human myoblast cultures and in humanized DMD (hDMD) mice with local delivery by i.m injection to identify PMOs of maximal skipping potency. Finally we selected 5 oligomers as vivo-PMOs targeting each of the exon 51 and 53 and examined their exon skipping efficiency in the hDMD mice by systemic delivery. We were able to identify PMOs with high exon skipping efficiency in all muscles. The selected PMOs will be further validated in patient-derived fibroblasts and then be applied to clinical trials for DMD treatment.

A series of low molecular weight polyethyleneimine (LPEI) conjugated Pluronic copolymers (PCMs) were synthesized and evaluated in C2C12 myoblasts and CHO cells in vitro and in dystrophic mdx mice in vivo as gene delivery carriers. Pluronics with different molecular weights (Mw) and hydrophilic–lipophilic-balance (HLB), and two LPEIs (Mw: 0.8k, 1.2k Da) as composition of PCMs have been synthesized, and the dynamics between the structures and properties were investigated. The conjugation efficiency of PEI ranged from 77.5–95.4% with relatively higher levels of PEI conjugation to the Pluronic size in PCMs, with the Pluronics of smaller size achieving relatively higher levels of PEI conjugation. Almost all of the PCM polymers were able to bind and condense plasmid DNA effectively into particles of approximately 200 nm in solution at the polymer/DNA ratio of 2 and above. The PCMs composed of relatively moderate size (Mw: 2000–5000 Da), intermediate HLB (12–23) of Pluronics, and LPEI produce much higher gene delivery efficacy and less cytotoxicity as compared with PEI 25k in C2C12 myoblasts and CHO cells in vitro. The PCMs were also able to enhance gene delivery in mdx mice in vivo, indicating their potential as biocompatible gene delivery carriers.