Phosphorodiamidate Morpholino Oligomers Research Articles

Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino oligomers.

BackgroundAs the causative agent of COVID-19, SARS-CoV-2 is a pathogen of immense importance to global public health. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense compounds composed of a phosphorodiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking. MethodsFive PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5′-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method using a 48 h PPMO treatment time. Viral growth was measured with quantitative RT–PCR and TCID50 infectivity assays from experiments where cells received a 5 h PPMO treatment time.ResultsPPMO designed to base-pair with sequence in the 5′ terminal region or the leader transcription regulatory sequence region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titres by up to 4–6 log10 in cell cultures at 48–72 h post-infection, in a non-toxic and dose-responsive manner.ConclusionsThe data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further preclinical development.

Nonsequential Splicing Events Alter Antisense-Mediated Exon Skipping Outcome in COL7A1.

The COL7A1 gene encodes homotrimer fibrils essential for anchoring dermal and epidermal layers, and pathogenic mutations in COL7A1 can cause recessive or dominant dystrophic epidermolysis bullosa. As a monogenic disease gene, COL7A1 constitutes a potential target for antisense oligomer-mediated exon skipping, a therapy applicable to a growing number of other genetic disorders. However, certain characteristics of COL7A1: many exons, low average intron size, and repetitive and guanine-cytosine rich coding sequence, present challenges to the design of specific and effective antisense oligomers. While targeting COL7A1 exons 10 and 73 for excision from the mature mRNA, we discovered that antisense oligomers comprised of 2′-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers produced similar, but distinctive, splicing patterns including excision of adjacent nontargeted exons and/or retention of nearby introns in some transcripts. We found that the nonsequential splicing of certain introns may alter pre-mRNA processing during antisense oligomer-mediated exon skipping and, therefore, additional studies are required to determine if the order of intron removal influences multiexon skipping and/or intron retention in processing of the COL7A1 pre-mRNA.

DMD – THERAPY: P.288 Casimersen treatment in eligible patients with Duchenne muscular dystrophy: Safety, tolerability, and pharmacokinetics over 144 weeks of treatment

Casimersen is an investigational phosphorodiamidate morpholino oligomer designed for exon 45 skipping, which, in patients affected by that mutation, would result in the expression of a truncated, yet functional dystrophin protein. The objective of this randomized, phase 1/2 trial was to evaluate the safety, tolerability, and pharmacokinetics of casimersen in male, nonambulatory (incapable of walking ≥300 meters on the 6-minute walking test [6-MWT]) patients with DMD, aged 7–21 years, with mutations amenable to exon 45 skipping. Patients were randomized 2:1 to receive double-blind, intravenous casimersen in increasing doses at ≥2-week intervals (4, 10, 20, and 30 mg/kg/week) or placebo, for 12 weeks; all patients then received open-label casimersen 30 mg/kg for up to 132 weeks. Twelve patients were enrolled (casimersen, n=8; placebo, n=4), with the mean age of 13.6 years and the mean 6-MWT distance of 39 meters. Eleven (92%) patients completed the study (casimersen-casimersen, n=8; placebo-casimersen, n=3). All patients experienced ≥1 treatment-emergent adverse event (TEAE). In the double-blind period, most TEAEs in casimersen-treated patients (159/175; 91%) and all TEAEs in placebo-treated patients (11/11) were mild in severity. Three casimersen-treated patients experienced five serious AEs, which were considered not related to treatment and resolved during the study. No patient discontinued the trial due to TEAEs. No patterns, trends, or abnormalities were observed in clinical laboratory parameters. All pharmacokinetic parameters for casimersen 30 mg/kg/week at Weeks 7 and 60, including concentration vs time profiles, were similar. In conclusion, casimersen was well tolerated in patients with DMD amenable to exon 45 skipping, with a pharmacokinetic profile that suggests little to no accumulation following weekly dosing at 30 mg/kg.

DMD – THERAPY: P.283 Long-term safety and efficacy of golodirsen in male patients with Duchenne muscular dystrophy amenable to Exon 53 skipping

Golodirsen is a synthetic phosphorodiamidate morpholino oligomer designed to restore the mRNA reading frame in patients with DMD gene mutations amenable to exon 53 skipping. We report the safety and efficacy of long-term golodirsen treatment in this first-in-human, phase 1/2, two-part, multicenter trial. Twelve patients from part 1 (randomized, 12-week dose-titration phase; golodirsen [n = 8], placebo [n = 4]) plus 13 additional patients with genotypically confirmed DMD amenable to exon 53 skipping therapy were included in part 2 (open label).

Skipping Exon-v6 from CD44v6-Containing Isoforms Influences Chemotherapy Response and Self-Renewal Capacity of Gastric Cancer Cells.

De novo expressed CD44 isoforms containing exon-v6 are frequently associated with gastric cancer (GC) aggressiveness, and may predict chemotherapy response in vitro. Whether exon-v6 itself is responsible for conferring these properties to CD44v6-containing isoforms remains to be elucidated. CRISPR/Cas9 and Phosphorodiamidate Morpholino oligomers (PMOs) were used to induce specific exon-v6 skipping, maintaining the CD44 reading frame, in two GC cell lines endogenously expressing CD44v6. Cisplatin and 5-fluorouracil treatment response, and self-renewal ability was compared between CRISPR/Cas9-edited, CD44v6 knockdown and mock cells. We obtained homozygous genome-edited cell lines with exon-v6 deletion. Edited cells transcribed CD44v isoforms presenting in frame v5–v7 splicing, mimicking exon-v6 skipping. Results showed that removing specifically exon-v6 sensitizes cells to cisplatin and impairs cells’ self-renewal ability, similarly to CD44v6 knockdown. In parallel, we also tested a clinically feasible approach for transient exon-v6 skipping with a PMO-based strategy. We demonstrate that exon-v6 specific removal from CD44v isoforms increases cell sensitivity to cisplatin and impairs GC cells self-renewal. We trust that a PMO approach designed towards CD44v6 overexpressing GC cells may be a suitable approach to sensitize tumor cells for conventional therapy.

Morpholino Oligomer-Induced Dystrophin Isoforms to Map the Functional Domains in the Dystrophin Protein

Dystrophin plays a crucial role in maintaining sarcolemma stability during muscle contractions, and mutations that prevent the expression of a functional protein cause Duchenne muscular dystrophy (DMD). Antisense oligonucleotide-mediated manipulation of pre-messenger RNA splicing to bypass Duchenne-causing mutations and restore functional dystrophin expression has entered the clinic for the most common DMD mutations. The rationale of “exon skipping” is based upon genotype-phenotype correlations observed in Becker muscular dystrophy, a milder allelic disorder generally characterized by in-frame deletions and internally truncated but semi-functional dystrophin isoforms. However, there is a lack of genotype-phenotype correlations downstream of DMD exon 55, as deletions in this region are rare and most single exon deletions would disrupt the reading frame. Consequently, the amenability of mutations in this region of the DMD gene to exon skipping strategies remains unknown. Here, we induced “Becker muscular dystrophy-like” in-frame dystrophin isoforms in vivo by intraperitoneal injection of peptide-conjugated phosphorodiamidate morpholino oligomers targeting selected exons. The dystrophin isoform encoded by the transcript lacking exons 56+57 appears to be more functional than that encoded by the 58+59-deleted transcript, as determined by higher dystrophin expression, stabilized β-dystroglycan, and less severe dystrophic pathology, indicating some potential for the strategy to address Duchenne-causing mutations affecting these exons.

Single Exon Skipping Can Address a Multi-Exon Duplication in the Dystrophin Gene.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease typically caused by protein-truncating mutations that preclude synthesis of a functional dystrophin. Exonic deletions are the most common type of DMD lesion, however, whole exon duplications account for between 10–15% of all reported mutations. Here, we describe in vitro evaluation of antisense oligonucleotide-induced splice switching strategies to re-frame the transcript disrupted by a multi-exon duplication within the DMD gene. Phosphorodiamidate morpholino oligomers and phosphorodiamidate morpholino oligomers coupled to a cell penetrating peptide were evaluated in a Duchenne muscular dystrophy patient cell strain carrying an exon 14–17 duplication. Two strategies were employed; the conventional approach was to remove both copies of exon 17 in addition to exon 18, and the second strategy was to remove only the first copy of exon 17. Both approaches result in a larger than normal but in-frame DMD transcript, but surprisingly, the removal of only the first exon 17 appeared to be more efficient in restoring dystrophin, as determined using western blotting. The emergence of a normal sized DMD mRNA transcript that was not apparent in untreated samples may have arisen from back splicing and could also account for some of the dystrophin protein being produced.

Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping

Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene. Among various treatments available for DMD, antisense oligonucleotides (ASOs) mediated exon skipping is a promising therapeutic approach. For successful treatments, however, it is requisite to rigorously optimise oligonucleotide chemistries as well as chemical modifications of ASOs. To achieve this, here, we aim to develop a novel enhanced green fluorescence protein (EGFP)-based reporter assay system that allows us to perform efficient and high-throughput screenings for ASOs. We design a new expression vector with a CAG promoter to detect the EGFP fluorescence only when skipping of mdx-type exon 23 is induced by ASOs. Then, an accurate screening was successfully conducted in C57BL/6 primary myotubes using phosphorodiamidate morpholino oligomer or locked nucleic acids (LNA)/2′-OMe mixmers with different extent of LNA inclusion. We accordingly generated a novel transgenic mouse model with this EGFP expression vector (EGFP-mdx23 Tg). Finally, we confirmed that the EGFP-mdx23 Tg provided a highly sensitive platform to check the effectiveness as well as the biodistribution of ASOs for exon skipping therapy. Thus, the assay system provides a simple yet highly sensitive platform to optimise oligonucleotide chemistries as well as chemical modifications of ASOs.

Splice modulating antisense oligonucleotides restore some acid-alpha-glucosidase activity in cells derived from patients with late-onset Pompe disease

Pompe disease is caused by mutations in the GAA gene, resulting in deficient lysosomal acid-α-glucosidase activity in patients, and a progressive decline in mobility and respiratory function. Enzyme replacement therapy is one therapeutic option, but since not all patients respond to this treatment, alternative interventions should be considered. One GAA mutation, c.-32-13T > G, impacts upon normal exon 2 splicing and is found in two-thirds of late-onset cases. We and others have explored a therapeutic strategy using splice modulating phosphorodiamidate morpholino oligomers to enhance GAA exon 2 inclusion in the mature mRNA of patients with one c.-32-13T > G allele. We designed 20 oligomers and treated fibroblasts derived from five patients to identify an oligomer sequence that maximally increased enzyme activity in all fibroblasts. The most effective splice correcting oligomer was chosen to treat forced-myogenic cells, derived from fibroblasts from nine patients carrying the c.-32-13T > G mutation. After transfection, we show increased levels of the full-length GAA transcript, acid-α-glucosidase protein, and enzyme activity in all patients’ myogenic cells, regardless of the nature of the mutation in the other allele. This data encourages the initiation of clinical trials to assess the therapeutic efficacy of this oligomer for those patients carrying the c.-32-13T > G mutation.

Transient silencing of transforming growth factor‐β1 expression restores nitric oxide generation in diabetic hematopoietic stem/progenitor cells: Role of thrombospondin‐1.

Adult hematopoietic stem/progenitor cells (HSPCs) participate in the cardiovascular repair by stimulating regeneration and angiogenic functions of endothelium. Long‐term diabetes is associated with impaired vasoprotective functions of HSPCs. Transforming growth factor β (TGFβ1) is pleiotropic regulator of HSPC functions. Previous studies have shown that transient silencing of TGFβ1 expression improves in vivo migratory functions of diabetic HSPCs partly by restoring nitric oxide (NO) generation. This study tested the hypothesis that restoration of NO by TGFβ1‐silencing is mediated by thrombospondin‐1 (TSP1)/CD47 pathway. TSP1 is known to attenuate NO generation via inhibition of endothelial nitric oxide synthase (eNOS). HSPCs were isolated from peripheral blood samples obtained from either male or female healthy (n=25) or diabetic (both type 1 and type 2) (n=27) individuals (50–80 years of age) by immunomagnetic enrichment. TGFβ1 expression was transiently blocked by using TGFβ1‐antisense delivered in the form of phosphorodiamidate morpholino oligomer (PMO‐TGFβ1). TGFβ1 and TSP1 gene expressions were determined in HSPCs treated with either PMO‐control or PMO‐TGFβ1. NO generation induced by stromal‐derived factor‐1α (SDF) was determined by DAF‐FM flow cytometry. Diabetic cells have higher expression of TGFβ1 or TSP1 compared to that observed in cells derived from healthy individuals (n=10). Treatment with TGFβ1‐PMO decreased the expression of TGFβ1, which was associated with decreased TSP1 expression. CD47 expression is similar in healthy or diabetic individuals. NO generation by SDF is attenuated in diabetic compared to healthy cells (P<0.01, n=5). PMO‐TGFβ1‐treated diabetic cells showed increased generation of NO in response to SDF compared to PMO‐control‐treatment (P<0.01, n=5). Future studies will determine the effect of TSP1 on SDF/NO generation in cells with or without CD47‐siRNA, to further support the hypothesis. Collectively, these studies indicate the involvement of TGFβ1/TSP1/CD47/eNOS pathway in diabetic impairment of NO generation however involvement of other pathways cannot be ruled out.Support or Funding InformationThis study is partly supported by funding from NIH National Institute of Aging (AG056681).

Combined Treatment with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomer-PPMO and AAV-U7 Rescues the Severe DMD Phenotype in Mice

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by an absence of the dystrophin protein, which is essential for muscle fiber integrity. Among the developed therapeutic strategies for DMD, the exon-skipping approach corrects the frameshift and partially restores dystrophin expression. It could be achieved through the use of antisense sequences, such as peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) or the small nuclear RNA-U7 carried by an adeno-associated virus (AAV) vector. AAV-based gene therapy approaches have potential for use in DMD treatment but are subject to a major limitation: loss of the AAV genome, necessitating readministration of the vector, which is not currently possible, due to the immunogenicity of the capsid. The PPMO approach requires repeated administrations and results in only weak cardiac dystrophin expression. Here, we evaluated a combination of PPMO- and AAV-based therapy in a mouse model of severe DMD. Striking benefits of this combined therapy were observed in striated muscles, with marked improvements in heart and diaphragm structure and function, with unrivalled extent of survival, opening novel therapeutic perspectives for patients.

Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle.

The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.

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

BackgroundThe 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.ResultsTAPs 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.ConclusionThe 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.

Golodirsen for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a life-shortening X-linked genetic disorder characterized by progressive wasting and weakening of muscles in boys. Loss-of-function mutations in the DMD gene, which codes for dystrophin, lead to this disease. The majority of mutations in this gene result in the exclusion of one or more exons from the transcript, eventually causing the remaining exons not to fit together correctly (i.e., out-of-frame mutations). Antisense oligonucleotides, e.g., phosphorodiamidate morpholino oligomers (PMOs), can induce therapeutic exon skipping during pre-mRNA processing to restore the reading frame of the primary transcript of DMD. As a result, truncated but partially functional dystrophin is produced, potentially slowing down the disease progression. Golodirsen is a provisionally approved PMO-based drug for approx. 8% of all DMD patients amenable to exon 53 skipping. This article summarizes golodirsen's pharmacology, efficacy and safety information. It also discusses some controversies that golodirsen met after the approval.

Detection of Locked Nucleic Acid Gapmers from Mouse Muscle Samples Using ELISA.

Antisense oligonucleotide (ASO)-mediated therapy is promising for the treatment of a variety of genetic disorders, suchas Duchenne muscular dystrophy. As more ASOs advance in therapeutic development and enter clinical trials, it becomes necessary to have a means of quantifying their amounts in biological samples post-treatment. This information will be valuable for evaluating the safety and pharmacokinetic profiles of ASOs, and in deciding how the efficacy of these drugs can be improved. Gapmers are a class of ASOs characterized by having a central DNA portion that is surrounded by chemically modified nucleotides on both ends. While relativelysimpleand accessiblemethods to quantify other ASOs such as phosphorodiamidate morpholino oligomers (PMOs)using enzyme-linked immunosorbent assay (ELISA)-based techniques are available and have been used for in vivo studies, nosuch method is available for gapmers to our knowledge. Here, we describe a sensitive ELISA protocol that can be used to quantify the levels of locked nucleic acid (LNA) gapmers in mouse muscle tissue.

Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing.

Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5′ end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.

Amelioration of intracellular Ca2+ regulation by exon-45 skipping in Duchenne muscular dystrophy-induced pluripotent stem cell-derived cardiomyocytes

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by frameshift mutations in the DMD gene. DMD involves cardiac muscle, and the presence of ventricular arrhythmias or congestive failure is critical for prognosis. Several novel therapeutic approaches are being evaluated in ongoing clinical trials. Among them, exon-skipping therapy to correct frameshift mutations with antisense oligonucleotides is promising; however, their therapeutic efficacies on cardiac muscle in vivo remain unknown. In this study, we established induced-pluripotent stem cells (iPSCs) from T cells from a DMD patient carrying a DMD-exon 46–55 deletion, differentiated the iPSCs into cardiomyocytes, and treated them with phosphorodiamidate morpholino oligomers. The efficiency of exon-45 skipping increased in a dose-dependent manner and enabled restoration of the DMD gene product, dystrophin. Further, Ca2+-imaging analysis showed a decreased number of arrhythmic cells and improved transient Ca2+ signaling after exon skipping. Thus, exon-45 skipping may be effective for cardiac involvement in DMD patients harboring the DMD-exon 46–55 deletion.

Perspectives towards antiviral drug discovery against Ebola virus.

Ebola virus disease (EVD), caused by Ebola viruses, resulted in more than 11 500 deaths according to a recent 2018 WHO report. With mortality rates up to 90%, it is nowadays one of the most deadly infectious diseases. However, no Food and Drug Administration‐approved Ebola drugs or vaccines are available yet with the mainstay of therapy being supportive care. The high fatality rate and absence of effective treatment or vaccination make Ebola virus a category‐A biothreat pathogen. Fortunately, a series of investigational countermeasures have been developed to control and prevent this global threat. This review summarizes the recent therapeutic advances and ongoing research progress from research and development to clinical trials in the development of small‐molecule antiviral drugs, small‐interference RNA molecules, phosphorodiamidate morpholino oligomers, full‐length monoclonal antibodies, and vaccines. Moreover, difficulties are highlighted in the search for effective countermeasures against EVD with additional focus on the interplay between available in silico prediction methods and their evidenced potential in antiviral drug discovery.

P.141PPMO-mediated skipping therapy of duplicated exon 2 in the DMD gene

Duchenne muscular dystrophy (DMD) is caused by fatal mutations that disrupt the reading frame of the DMD gene, leading to lack of dystrophin protein. The only approved gene corrective therapy is an antisense phosphorodiamidate morpholino oligomer (PMO) that alters splicing to exclude exon 51 from the mRNA, restoring translation of a shortened but functionally active protein. Use of antisense PMO skipping in the presence of single exon duplications, in contrast, may restore a wild-type DMD transcript and dystrophin protein. Here we report the application of cell-penetrating peptide-conjugated PMO, or PPMO, to target the DMD exon 2 splice acceptor site in Dup2 mice, which carry a duplication of exon 2 (the most common single duplication mutation in DMD patients, accounting for around 10% of all duplication mutations). In the presence of an exon 2 duplication, skipping can result in two therapeutic transcripts. Skipping of a single copy of exon 2 results in a wild-type transcript, whereas skipping both copies results in a transcript lacking exon 2 entirely (Del2), but from which functionally active dystrophin protein can be expressed via utilization of an exon 5 internal ribosome entry site (IRES). A single intravenous injection of PPMO was delivered to 12 week old mice at three dose levels; saline injection and scrambled PPMO were used as controls. All mice (n=5-6 each) were sacrificed one week post-injection. Five muscles–tibialis anterior (TA), gastrocnemius (Gas), quadriceps (Qua), diaphragm (Dia) and heart were harvested for analysis of DMD mRNA transcripts by RT-PCR, and for dystrophin protein expression by western blot and immunofluorescence staining. The single injection resulted in a clear dose response of exon skipping, maximal at the highest dose tested; robust skipping of the duplicated exon 2 (both WT and Del2 transcripts) was seen in skeletal muscles, including TA (40.8%), Gas (39.7%), Qua (53.5%), and Dia (53.1%), with minimal skipping observed in heart muscle (8%). Dystrophin protein restoration was seen at levels of up to 13% in Dia and up to 15% in Gas, and at lower levels in other tissues, likely reflecting the single injection and short time period allowed for expression. We are evaluating longer term expression, with a goal of developing a meaningful therapy for patients with exon 2 duplications and potentially other 5′ DMD mutations.

Hexose Potentiates Peptide-Conjugated Morpholino Oligomer Efficacy in Cardiac Muscles of Dystrophic Mice in an Age-Dependent Manner

Insufficient delivery of oligonucleotides to muscle and heart remains a barrier for clinical implementation of antisense oligonucleotide (AO)-mediated exon-skipping therapeutics in Duchenne muscular dystrophy (DMD), a lethal monogenic disorder caused by frame-disrupting mutations in the DMD gene. We previously demonstrated that hexose, particularly an equal mix of glucose:fructose (GF), significantly enhanced oligonucleotide delivery and exon-skipping activity in peripheral muscles of mdx mice; however, its efficacy in the heart remains limited. Here we show that co-administration of GF with peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO, namely, BMSP-PMO) induced an approximately 2-fold higher level of dystrophin expression in cardiac muscles of adult mdx mice compared to BMSP-PMO in saline at a single injection of 20 mg/kg, resulting in evident phenotypic improvement in dystrophic mdx hearts without any detectable toxicity. Dystrophin expression in peripheral muscles also increased. However, GF failed to potentiate BMSP-PMO efficiency in aged mdx mice. These findings demonstrate that GF is applicable to both PMO and PPMO. Furthermore, GF potentiates oligonucleotide activity in mdx mice in an age-dependent manner, and, thus, it has important implications for its clinical deployment for the treatment of DMD and other muscular disorders.