Sapana N. Shah

Exon skipping restores dystrophin expression, but fails to prevent disease progression in later stage dystrophic dko mice.

Antisense therapy with both chemistries of phosphorodiamidate morpholino oligomers (PMOs) and 2′-O-methyl phosphorothioate has demonstrated the capability to induce dystrophin expression in Duchenne muscular dystrophy (DMD) patients in phase II-III clinical trials with benefit in muscle functions. However, potential of the therapy for DMD at different stages of the disease progression is not understood. In this study, we examined the effect of peptide-conjugated PMO (PPMO)-mediated exon skipping on disease progression of utrophin-dystrophin-deficient mice (dko) of four age groups (21–29, 30–39, 40–49 and 50+ days), representing diseases from early stage to advanced stage with severe kyphosis. Biweekly intravenous (i.v.) administration of the PPMO restored the dystrophin expression in nearly 100% skeletal muscle fibers in all age groups. This was associated with the restoration of dystrophin-associated proteins including functional glycosylated dystroglycan and neuronal nitric synthase. However, therapeutic outcomes clearly depended on severity of the disease at the time the treatment started. The PPMO treatment alleviated the disease pathology and significantly prolonged the life span of the mice receiving treatment at younger age with mild phenotype. However, restoration of high levels of dystrophin expression failed to prevent disease progression to the mice receiving treatment when disease was already at advanced stage. The results could be critical for design of clinical trials with antisense therapy to DMD.

Pluronic–PEI copolymers enhance exon-skipping of 2′- O -methyl phosphorothioate oligonucleotide in cell culture and dystrophic mdx mice

A series of small-size polyethylenimine (PEI)-conjugated pluronic polycarbamates (PCMs) have been investigated for the ability to modulate the delivery of 2′-O-methyl phosphorothioate RNA (2′-OMePS) in vitro and in dystrophic mdx mice. The PCMs retain strong binding capacity to negatively charged oligomer as demonstrated by agarose gel retardation assay, with the formation of condensed polymer/oligomer complexes at a wide-range weight ratio from 1:1 to 20:1. The condensed polymer/oligomer complexes form 100–300 nm nanoparticles. Exon-skipping effect of 2′-OMePS was dramatically enhanced with the use of the most effective PCMs in comparison with 2′-OMePS alone in both cell culture and in vivo, respectively. More importantly, the effective PCMs, especially those composed of moderate size (2k–5kDa) and intermediate hydrophilic–lipophilic balance (7–23) of pluronics, enhanced exon-skipping of 2′-OMePS with low toxicity as compared with Lipofectamine-2000 in vitro or PEI 25k in vivo. The variability of individual PCM for delivery of antisense oligomer and plasmid DNA indicate the complexity of interaction between polymer and their cargos. Our data demonstrate the potential of PCMs to mediate delivery of modified antisense oligonucleotides to the muscle for treating muscular dystrophy or other appropriate myodegenerative diseases.

Synthesis and Evaluation of Tween 85-LPEI Copolymers for GeneTransfection In vitro and In vivo

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.

Long-Term Treatment of Tamoxifen and Raloxifene Alleviates Dystrophic Phenotype and Enhances Muscle Functions of FKRP Dystroglycanopathy

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.

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.

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.

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

Abstract 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.

In Vivo Evaluation of Dystrophin Exon Skipping in mdx Mice

Dystrophin exon skipping in mdx mice has been the key model for the development of antisense therapy in vivo. Evaluation of exon skipping in this model involves the following two aspects: (1) efficiency and accuracy of exon skipping and levels of dystrophin expression determined by RT-PCR, immunochemistry, and western blotting; (2) therapeutic effects on muscle pathology and functions assessed by histology and functional assays including grip strength measurement, treadmill test, echocardiogram, and hemodynamics for cardiac functions. Here we describe some key considerations and the essential methodologies in detail for exon skipping in mdx mice.

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.

148. Screening and Optimization of Antisense Oligonucleotide for Skipping Human Dystrophin Exon 51 and 53

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.