An alternative strategy to increasing influenza virus replication for vaccine production in chicken embryo fibroblast (DF-1) cells by inhibiting interferon alpha and beta using peptide-conjugated phosphorodiamidate morpholino oligomers.

Introduction. Influenza is a global health issue causing substantial health and economic burdens on affected populations. Routine, annual vaccination for influenza virus is recommended for all persons older than 6 months of age. The propagation of the influenza virus for vaccine production is predominantly through embryonated chicken eggs.Hypothesis/Gap Statement. Many challenges face the propagation of the virus, including but not limited to low yields and lengthy production times. The development of a method to increase vaccine production in eggs or cell lines by suppressing cellular gene expression would be helpful to overcome some of the challenges facing influenza vaccine production.Aims. This study aimed to increase influenza virus titres by using a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), an antisense molecule, to suppress protein expression of the host genes interferon alpha (IFN-α) and interferon beta (IFN-ß) in chicken embryo fibroblast (DF-1) cells.Methods. The toxicity of PPMOs was evaluated by cytotoxicity assays, and their specificity to inhibit IFN-α and IFN-ß proteins was measured by ELISA. We evaluated the potential of anti-IFN-α and anti-IFN-ß PPMOs to reduce the antiviral proteins in influenza virus-infected DF-1 cells and compared the virus titres to untreated controls, nonsense-PPMO and JAK/STAT inhibitors. The effects of complementation and reconstitution of IFN-α and IFN-ß proteins in PPMO-treated-infected cells were evaluated, and the virus titres were compared between treatment groups.Results. Suppression of IFN-α by PPMO resulted in significantly reduced levels of IFN-α protein in treated wells, as measured by ELISA and was shown to not have any cytotoxicity to DF-1 cells at the effective concentrations tested. Treatment of the self-directing PPMOs increased the ability of the influenza virus to replicate in DF-1 cells. Over a 2-log10 increase in viral production was observed in anti-IFN-α and IFN-ß PPMO-treated wells compared to those of untreated controls at the initial viral input of 0.1 multiplicity of infection. The data from complementation and reconstitution of IFN-α and IFN-ß proteins in PPMO-treated-infected cells was about 82 and 97% compared to the combined PPMO-treated but uncomplemented group and untreated group, respectively. There was a 0.5-log10 increase in virus titre when treated with anti-IFN-α and IFN-ß PPMO compared to virus titre when treated with JAK/STAT inhibitors.Conclusions. This study emphasizes the utility of PPMO in allowing cell cultures to produce increased levels of influenza for vaccine production or alternatively, as a screening tool to cheaply test targets prior to the development of permanent knockouts of host gene expression.

Clinical, immunohistochemical, and genetic characterization of splice-altering biallelic DES variants: Therapeutic implications.

Pathogenic variants in the DES gene clinically manifest as progressive skeletal muscle weakness, cardiomyopathy with associated severe arrhythmias, and respiratory insufficiency, and are collectively known as desminopathies. While most DES pathogenic variants act via a dominant mechanism, recessively acting variants have also been reported. Currently, there are no effective therapeutic interventions for desminopathies of any type. Here, we report an affected individual with rapidly progressive dilated cardiomyopathy, requiring heart transplantation at age 13 years, in the setting of childhood-onset skeletal muscle weakness. We identified biallelic DES variants (c.640-13 T>A and c.1288+1 G>A) and show aberrant DES gene splicing in the affected individual's muscle. Through the generation of an inducible lentiviral system, we transdifferentiated fibroblast cultures derived from the affected individual into myoblasts and validated this system using RNA sequencing. We tested rationally designed, custom antisense oligonucleotides to screen for splice correction in these transdifferentiated cells and a functional minigene splicing assay. However, rather than correctly redirecting splicing, we found them to induce undesired exon skipping. Our results indicate that, while an individual precision-based molecular therapeutic approach to splice-altering pathogenic variants is promising, careful preclinical testing is imperative for each novel variant to test the feasibility of this type of approach for translation.

Electrical impedance myography detects dystrophin-related muscle changes in mdx mice.

BACKGROUND: The lack of functional dystrophin protein in Duchenne muscular dystrophy (DMD) causes chronic skeletal muscle inflammation and degeneration. Therefore, the restoration of functional dystrophin levels is a fundamental approach for DMD therapy. Electrical impedance myography (EIM) is an emerging tool that provides noninvasive monitoring of muscle conditions and has been suggested as a treatment response biomarker in diverse indications. Although magnetic resonance imaging (MRI) of skeletal muscles has become a standard measurement in clinical trials for DMD, EIM offers distinct advantages, such as portability, user-friendliness, and reduced cost, allowing for remote monitoring of disease progression or response to therapy. To investigate the potential of EIM as a biomarker for DMD, we compared longitudinal EIM data with MRI/histopathological data from an X-linked muscular dystrophy (mdx) mouse model of DMD. In addition, we investigated whether EIM could detect dystrophin-related changes in muscles using antisense-mediated exon skipping in mdx mice. METHODS: The MRI data for muscle T2, the magnetic resonance spectroscopy (MRS) data for fat fraction, and three EIM parameters with histopathology were longitudinally obtained from the hindlimb muscles of wild-type (WT) and mdx mice. In the EIM study, a cell-penetrating peptide (Pip9b2) conjugated antisense phosphorodiamidate morpholino oligomer (PPMO), designed to induce exon-skipping and restore functional dystrophin production, was administered intravenously to mdx mice. RESULTS: MRI imaging in mdx mice showed higher T2 intensity at 6 weeks of age in hindlimb muscles compared to WT mice, which decreased at ≥ 9 weeks of age. In contrast, EIM reactance began to decline at 12 weeks of age, with peak reduction at 18 weeks of age in mdx mice. This decline was associated with myofiber atrophy and connective tissue infiltration in the skeletal muscles. Repeated dosing of PPMO (10 mg/kg, 4 times every 2 weeks) in mdx mice led to an increase in muscular dystrophin protein and reversed the decrease in EIM reactance. CONCLUSIONS: These findings suggest that muscle T2 MRI is sensitive to the early inflammatory response associated with dystrophin deficiency, whereas EIM provides a valuable biomarker for the noninvasive monitoring of subsequent changes in skeletal muscle composition. Furthermore, EIM reactance has the potential to monitor dystrophin-deficient muscle abnormalities and their recovery in response to antisense-mediated exon skipping.

In Vivo Evaluation of Exon 51 Skipping in hDMD/Dmd-null Mice.

Duchenne muscular dystrophy (DMD) is a fatal X-linked condition that affects 1 in 3500-6000 newborn boys a year. An out-of-frame mutation in the DMD gene typically causes the condition. Exon skipping therapy is an emerging approach that uses antisense oligonucleotides (ASOs), short synthetic DNA-like molecules that can splice out mutated or frame-disrupting mRNA fragments, to restore the reading frame. The restored reading frame will be in-frame and will produce a truncated, yet functional protein. ASOs called phosphorodiamidate morpholino oligomers (PMO), including eteplirsen, golodirsen, and viltolarsen, have recently been approved by the US Food and Drug Administration as the first ASO-based drugs for DMD. ASO-facilitated exon skipping has been extensively studied in animal models. An issue that arises with these models is that the DMD sequence differs from the human DMD sequence. A solution to this issue is to use double mutant hDMD/Dmd-null mice, which only carry the human DMD sequence and are null for the mouse Dmd sequence. Here, we describe intramuscular and intravenous injections of an ASO to skip exon 51 in hDMD/Dmd-null mice, and the evaluation of its efficacy in vivo.

A cell-penetrating peptide enhances delivery and efficacy of phosphorodiamidate morpholino oligomers in mdx mice.

Antisense RNA technology is a strategy for the treatment of Duchenne muscular dystrophy (DMD), a progressive and universally fatal X-linked neuromuscular disease caused by frameshift mutations in the gene encoding dystrophin. Phosphorodiamidate morpholino oligomers (PMOs) are an antisense RNA platform that is used clinically in patients with DMD to facilitate exon skipping and production of an internally truncated, yet functional, dystrophin protein. Peptide-conjugated PMOs (PPMOs) are a next-generation platform in which a cell-penetrating peptide is conjugated to the PMO backbone, with the goal of increasing cellular uptake. RC-1001 is a PPMO that contains a proprietary cell-penetrating peptide and targets the Dmd mutation in mdx mice. It was evaluated in mdx mice for exon 23 skipping, dystrophin production, and functional efficacy. Single-dose RC-1001 dose dependently increased exon skipping and dystrophin protein levels in striated muscle and is associated with improvements in muscle function. Dystrophin protein levels were durable for 60 days. Three doses, each given 1 month apart, increased exon skipping to 99% in quadriceps and 43% in heart, with dystrophin protein levels at 39% and 9% of wild type, respectively. These findings support clinical development of PPMO therapies for the treatment of DMD.

Colorimetric aptasensor for sensitive detection of quinclorac based on exonuclease III-assisted cyclic release of phosphorodiamidate morpholino oligomer mimic enzyme strategy.

Here, a colorimetric aptasensor was constructed for sensitively detecting quinclorac (QNC), a common herbicide. The aptasensor involved a novel amplification strategy and a classical strand displacement strategy. The amplification strategy, termed exonuclease III (Exo III)-assisted cyclic release of phosphorodiamidate morpholino oligomer (PMO) mimic enzyme strategy, was developed based on two new findings on PMO: 1) DNA hybridized with PMO could resist Exo III digestion; 2) a designed G-rich PMO (named P2) could bind to hemin to form a G-quadruplex PMOzyme with peroxidase-like activity. In this strategy, a designed DNA-PMO duplex (D1-P1) completely hybridized with DNA2 (D2) in the other designed DNA-PMO duplex (D2-P2) to trigger D2 degradation by Exo III and cyclic release of P2. After that, the hemin-binding P2 catalyzed colorless tetra-methyl benzidine (TMB) to blue TMB+. The cycle process was performed at high Exo III concentrations without strict control and with constant background signals. In that case, the developed strategy was sensitive, efficient, easy to operate, reliable, and ultralow background. Meanwhile, a QNC aptamer was used to develop the strand displacement strategy based on magnetic beads. The colorimetric aptasensor was sensitive and selective for QNC detection with a detection limit of 7.1 ng mL-1. It was successfully applied to detect QNC in soil and river water with good recovery rates (92-98%) and a relative standard deviation (n = 3) <5%. The success of this study could provide a general reference strategy for developing sensitive aptasensors and other nucleic acid-related sensors.

Antisense Oligonucleotide Induction of the hnRNPA1b Isoform Affects Pre-mRNA Splicing of SMN2 in SMA Type I Fibroblasts.

Spinal muscular atrophy (SMA) is a severe, debilitating neuromuscular condition characterised by loss of motor neurons and progressive muscle wasting. SMA is caused by a loss of expression of SMN1 that encodes the survival motor neuron (SMN) protein necessary for the survival of motor neurons. Restoration of SMN expression through increased inclusion of SMN2 exon 7 is known to ameliorate symptoms in SMA patients. As a consequence, regulation of pre-mRNA splicing of SMN2 could provide a potential molecular therapy for SMA. In this study, we explored if splice switching antisense oligonucleotides could redirect the splicing repressor hnRNPA1 to the hnRNPA1b isoform and restore SMN expression in fibroblasts from a type I SMA patient. Antisense oligonucleotides (AOs) were designed to promote exon 7b retention in the mature mRNA and induce the hnRNPA1b isoform. RT-PCR and western blot analysis were used to assess and monitor the efficiency of different AO combinations. A combination of AOs targeting multiple silencing motifs in hnRNPA1 pre-mRNA led to robust hnRNPA1b induction, which, in turn, significantly increased expression of full-length SMN (FL-SMN) protein. A combination of PMOs targeting the same motifs also strongly induced hnRNPA1b isoform, but surprisingly SMN2 exon 5 skipping was detected, and the PMO cocktail did not lead to a significant increase in expression of FL-SMN protein. We further performed RNA sequencing to assess the genome-wide effects of hnRNPA1b induction. Some 3244 genes were differentially expressed between the hnRNPA1b-induced and untreated SMA fibroblasts, which are functionally enriched in cell cycle and chromosome segregation processes. RT-PCR analysis demonstrated that expression of the master regulator of these enrichment pathways, MYBL2 and FOXM1B, were reduced in response to PMO treatment. These findings suggested that induction of hnRNPA1b can promote SMN protein expression, but not at sufficient levels to be clinically relevant.

Pharmacological Profile of Viltolarsen for the Treatment of Duchenne Muscular Dystrophy: A Japanese Experience.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked recessive disorder characterized by progressive muscle loss and cardiorespiratory complications. Mutations in the DMD gene that eliminate the production of dystrophin protein are the underlying causes of DMD. Viltolarsen is a drug of phosphorodiamidate morpholino oligomer (PMO) chemistry, designed to skip exon 53 of the DMD gene. It aims to produce truncated but partially functional dystrophin in DMD patients and restore muscle function. Based on a preclinical study showing the ability of antisense PMOs targeting the DMD gene to improve muscle function in a large animal model, viltolarsen was developed by Nippon Shinyaku and the National Center of Neurology and Psychiatry in Japan. Following clinical trials conducted in Japan, Canada, and the United States showing significant improvements in muscle function, viltolarsen was approved for medical use in Japan in March 2020 and the United States in August 2020, respectively. Viltolarsen is a mutation-specific drug and will work for 8% of the persons with DMD who carry mutations amenable to exon 53 skipping. This review summarizes the pharmacological profile of viltolarsen, important clinical trials, and challenges, focusing on the contribution of Japanese patients and researchers in its development.

Antisense oligonucleotides: absorption, distribution, metabolism, and excretion.

INTRODUCTION: Antisense oligonucleotides (ASOs) have emerged as a promising novel drug modality that aims to address unmet medical needs. A record of six ASO drugs have been approved since 2016, and more candidates are in clinical development. ASOs are the most advanced class within the RNA-based therapeutics field. AREAS COVERED: This review highlights the two major backbones that are currently used to build the most advanced ASO platforms - the phosphorodiamidate morpholino oligomers (PMOs) and the phosphorothioates (PSs). The absorption, distribution, metabolism, and excretion (ADME) properties of the PMO and PS platforms are discussed in detail. EXPERT OPINION: Understanding the ADME properties of existing ASOs can foster further improvement of this cutting-edge therapy, thereby enabling researchers to safely develop ASO drugs and enhancing their ability to innovate. ABBREVIATIONS: 2'-MOE, 2'-O-methoxyethyl; 2'PS, 2 modified PS; ADME, absorption, distribution, metabolism, and excretion; ASO, antisense oligonucleotide; AUC, area under the curve; BNA, bridged nucleic acid; CPP, cell-penetrating peptide; CMV, cytomegalovirus; CNS, central nervous system; CYP, cytochrome P; DDI, drug-drug interaction; DMD, Duchenne muscular dystrophy; FDA, Food and Drug Administration; GalNAc3, triantennary N-acetyl galactosamine; IT, intrathecal; IV, intravenous; LNA, locked nucleic acid; mRNA, messenger RNA; NA, not applicable; PBPK, physiologically based pharmacokinetics; PD, pharmacodynamic; PK, pharmacokinetic; PMO, phosphorodiamidate morpholino oligomer; PMOplus, PMOs with positionally specific positive molecular charges; PPMO, peptide-conjugated PMO; PS, phosphorothioate; SC, subcutaneous; siRNA, small-interfering RNA; SMA, spinal muscular atrophy.

Safety, tolerability, and pharmacokinetics of casimersen in patients with Duchenne muscular dystrophy amenable to exon 45 skipping: A randomized, double-blind, placebo-controlled, dose-titration trial.

INTRODUCTION/AIMS: Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene resulting in the absence of dystrophin. Casimersen is a phosphorodiamidate morpholino oligomer designed to bypass frameshift DMD mutations and produce internally truncated, yet functional, dystrophin protein in patients amenable to exon 45 skipping. Our primary study objective was to evaluate safety and tolerability of casimersen; the secondary objective was to characterize the plasma pharmacokinetics. METHODS: This multicenter, phase 1/2 trial enrolled 12 participants (aged 7-21 years, who had limited ambulation or were nonambulatory) and comprised a 12-week, double-blind dose titration, then an open-label extension for up to 132 weeks. During dose titration, participants were randomized 2:1 to weekly casimersen infusions at escalating doses of 4, 10, 20, and 30 mg/kg (≥2 weeks per dose), or placebo. RESULTS: Participants received casimersen for a mean 139.6 weeks. Treatment-emergent adverse events (TEAEs) occurred in all casimersen- and placebo-treated participants and were mostly mild (over 91.4%) and unrelated to casimersen or its dose. There were no deaths, dose reductions, abnormalities in laboratory parameters or vital signs, or casimersen-related serious AEs. Casimersen plasma concentration increased with dose and declined similarly for all dose levels over 24 hours postinfusion. All pharmacokinetic parameters were similar at weeks 7 and 60. DISCUSSION: Casimersen was well tolerated in participants with DMD amenable to exon 45 skipping. Most TEAEs were mild, nonserious, and unrelated to casimersen. Plasma exposure was dose proportional with no suggestion of plasma accumulation. These results support further studies of casimersen in this population.

Antisense Oligonucleotide Treatment in a Humanized Mouse Model of Duchenne Muscular Dystrophy and Highly Sensitive Detection of Dystrophin Using Western Blotting.

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disorder affecting many children. The disease is caused by the lack of dystrophin production and characterized by muscle wasting. The most common causes of death are respiratory failure and heart failure. Antisense oligonucleotide-mediated exon skipping using a phosphorodiamidate morpholino oligomer (PMO) is a promising therapeutic approach for the treatment of DMD. In preclinical studies, dystrophic mouse models are commonly used for the development of therapeutic oligos. We employ a humanized model carrying the full-length human DMD transgene along with the complete knockout of the mouse Dmd gene. In this model, the effects of human-targeting AOs can be tested without cross-reaction between mouse sequences and human sequences (note that mdx, a conventional dystrophic mouse model, carries a nonsense point mutation in exon 23 and express the full-length mouse Dmd mRNA, which is a significant complicating factor). To determine if dystrophin expression is restored, the Western blotting analysis is commonly performed; however, due to the extremely large protein size of dystrophin (427 kDa), detection and accurate quantification of full-length dystrophin can be a challenge. Here, we present methodologies to systemically inject PMOs into humanized DMD model mice and determine levels of dystrophin restoration via Western blotting. Using a tris-acetate gradient SDS gel and semi-dry transfer with three buffers, including the Concentrated Anode Buffer, Anode Buffer, and Cathode Buffer, less than 1% normal levels of dystrophin expression are easily detectable. This method is fast, easy, and sensitive enough for the detection of dystrophin from both cultured muscle cells and muscle biopsy samples.

Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.

Developing DMD therapeutics: a review of the effectiveness of small molecules, stop-codon readthrough, dystrophin gene replacement, and exon-skipping therapies.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin (DMD) gene. Most patients die from respiratory failure or cardiomyopathy. There are significant unmet needs for treatments for DMD as the standard of care is principally limited to symptom relief through treatments including steroids. AREAS COVERED: This review summarizes safety and efficacy in promising areas of DMD therapeutics - small molecules, stop codon readthrough, gene replacement, and exon skipping - under clinical examination from 2015-2020 as demonstrated in the NIH Clinical Trials and PubMed search engines. EXPERT OPINION: Currently, steroids persist as the most accessible medicine for DMD. Stop-codon readthrough, gene replacement, and exon-skipping therapies all aim to restore dystrophin expression. Of these strategies, gene replacement therapy has recently gained momentum while exon-skipping retains great traction. The  FDA approval of three exon-skipping antisense oligonucleotides illustrate this regulatory momentum, though the effectiveness and sequence design of eteplirsen remain controversial. Cell-penetrating peptides promise to more efficaciously treat DMD-related cardiomyopathy.The recent success of antisense therapies, however, poses major regulatory challenges. To fully realize the benefits of exon-skipping, including cocktail oligonucleotide-mediated multiple exon-skipping and oligonucleotide drugs for very rare mutations, regulatory challenges need to be addressed in coordination with scientific advances.

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 ß-dystroglycan, and less severe dystrophic pathology, indicating some potential for the strategy to address Duchenne-causing mutations affecting these exons.

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.

Antisense targeting of CD47 enhances human cytotoxic T-cell activity and increases survival of mice bearing B16 melanoma when combined with anti-CTLA4 and tumor irradiation.

Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.

Identification of a Novel Deep Intronic Mutation in CAPN3 Presenting a Promising Target for Therapeutic Splice Modulation.

Calpainopathy, also known as limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) or LGMD R1 Calpain3-related, is one of the most common genetically characterized forms of limb-girdle muscular dystrophy with a wide range of phenotypic severity. We evaluated a consanguineous family with a clinical phenotype consistent with calpainopathy in whom conventional sequencing did not detect any mutations in the CAPN3 gene. Using whole exome sequencing paired with haplotype analysis, we identified a homozygous deep intronic single base pair deletion in CAPN3 (c.946-29delT). Familial segregation studies were consistent with recessive inheritance. Immunoblotting of muscle tissue from the patient showed complete absence of calpain 3. In silico analysis predicted the deletion to disrupt the branch point and subsequently alter splicing of exon 7. Studies of patient fibroblasts and muscle tissue confirmed altered splicing, resulting in an inclusion of a 389-bp intronic sequence upstream of exon 7, originating from a cryptic splice acceptor site in intron 6. This out-of-frame insertion results in a premature stop codon, leading to an apparent absence of protein likely due to degradation of the transcript via nonsense-mediated decay. We then designed phosphorodiamidate morpholino oligomers (PMOs) as splice modulators to block the new splice acceptor site. This approach successfully prevented the aberrant splicing - reverting the majority of the splice to the wildtype transcript. These results confirm the pathogenicity of this novel deep intronic mutation and provide a mutation-specific therapeutic strategy. Thus, deep intronic mutations in CAPN3 may be pathogenic and should be considered in the appropriate clinical setting. The identification of mutations which may be missed by traditional Sanger sequencing is essential as they may be excellent targets for individualized therapeutic strategies using RNA-directed splice modulation.

Orthogonal analysis of dystrophin protein and mRNA as a surrogate outcome for drug development.

Aim: Detection of drug-induced dystrophin in patient muscle biopsy is a surrogate outcome measure for Duchenne muscular dystrophy. We sought to establish and validate an orthogonal approach to measurement of dystrophin protein and RNA in muscle biopsies. Materials & methods: Validated methods were developed for dystrophin western blotting, mass spectrometry, immunostaining and reverse transcriptase PCR of biopsy mRNA using muscle biopsy standards. Results: Both western blotting and mass spectrometry validated methods demonstrated good linearity, and acceptable precision and accuracy with a lower limit of quantitation at 1%. Immunostaining and reverse transcriptase PCR methods were shown to be reliable. Conclusion: The described orthogonal approach is sufficient to support measures of dystrophin as a surrogate outcome in a clinical trial.

Exons 45-55 Skipping Using Mutation-Tailored Cocktails of Antisense Morpholinos in the DMD Gene.

Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45-55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45-55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45-55 skipping with removing 3, 8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45-55 skipping and treat over 65% of DMD patients carrying out-of-frame or in-frame deletions.

Current understanding and treatment of cardiac and skeletal muscle pathology in laminin-α2 chain-deficient congenital muscular dystrophy.

Congenital muscular dystrophy (CMD) is a class of severe early-onset muscular dystrophies affecting skeletal/cardiac muscles as well as the central nervous system (CNS). Laminin-α2 chain-deficient congenital muscular dystrophy (LAMA2 MD), also known as merosin-deficient congenital muscular dystrophy type 1A (MDC1A), is an autosomal recessive CMD characterized by severe muscle weakness and degeneration apparent at birth or in the first 6 months of life. LAMA2 MD is the most common congenital muscular dystrophy, affecting approximately 4 in 500,000 children. The most common cause of death in early-onset LAMA2 MD is respiratory tract infection, with 30% of them dying within the first decade of life. LAMA2 MD is caused by loss-of-function mutations in the LAMA2 gene encoding for the laminin-α2 chain, one of the subunits of laminin-211. Laminin-211 is an extracellular matrix protein that functions to stabilize the basement membrane and muscle fibers during contraction. Since laminin-α2 is expressed in many tissue types including skeletal muscle, cardiac muscle, Schwann cells, and trophoblasts, patients with LAMA2 MD experience a multi-systemic clinical presentation depending on the extent of laminin-α2 chain deficiency. Cardiac manifestations are typically associated with a complete absence of laminin-α2; however, recent case reports highlight cardiac involvement in partial laminin-α2 chain deficiency. Laminin-211 is also expressed in the brain, and many patients have abnormalities on brain imaging; however, mental retardation and/or seizures are rarely seen. Currently, there is no cure for LAMA2 MD, but various therapies are being investigated in an effort to lessen the severity of LAMA2 MD. For example, antisense oligonucleotide-mediated exon skipping and CRISPR-Cas9 genome editing have efficiently restored the laminin-α2 chain in mouse models in vivo. This review consolidates information on the clinical presentation, genetic basis, pathology, and current treatment approaches for LAMA2 MD.