Cardiac therapies for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating disease that results in life-limiting complications such as loss of skeletal muscle function as well as respiratory and cardiac complications. Advanced therapeutics in pulmonary care have significantly reduced respiratory complication-related mortality, making cardiomyopathy the main determinant factor of survival. While there are multiple therapies such as the use of anti-inflammatory drugs, physical therapy, and ventilatory assistance targeted toward delaying the disease progression in DMD, a cure remains elusive. In the last decade, several therapeutic approaches have been developed to improve patient survival. These include small molecule-based therapy, micro-dystrophin gene delivery, CRISPR-mediated gene editing, nonsense readthrough, exon skipping, and cardiosphere-derived cell therapy. Associated with the specific benefits of each of these approaches are their individual risks and limitations. The variability in the genetic aberrations leading to DMD also limits the widespread use of these therapies. While numerous approaches have been explored to treat DMD pathophysiology, only a handful have successfully advanced through the preclinical stages. In this review, we summarize the currently approved as well as the most promising therapeutics undergoing clinical trials aimed toward treating DMD with a focus on its cardiac manifestations.

Restoring Dystrophin Expression by Skipping Exons 6 and 8 in Neonatal Dystrophic Dogs.

Duchenne muscular dystrophy (DMD) is caused by the mutations in the DMD gene resulting in no dystrophin production. Skipping DMD exons using phosphorodiamidate morpholino oligomers (PMOs) is an emerging treatment strategy that can restore the reading frame of the mutated gene and produce truncated but functional dystrophin protein. To date, four PMOs, including eteplirsen, casimersen, viltolarsen, and golodirsen, have been conditionally approved by the FDA for the treatment of DMD. Since degeneration of muscle fibers and irreversible fibrosis occur from childhood, the earlier treatment is preferred. The canine X-linked muscular dystrophy in Japan (CXMDj), a dog model of DMD, produces no dystrophin and exhibits a severe phenotype similar to human patients from early childhood. As such, CXMDj, which harbors a splice site mutation in intron 6, is a useful model for examining the long-term effects of early PMO treatment. In this chapter, we describe the systemic delivery of a cocktail of four PMOs that can successfully induce multiple exon skipping (exons 6-9) in neonatal dystrophic dogs. We also describe the procedures to evaluate the efficacy and toxicity, including clinical grading of dystrophic dogs, ELISA-based quantification of PMOs, histology, RT-PCR, and western blotting.

Development of DG9 peptide-conjugated single- and multi-exon skipping therapies for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is primarily caused by out-of-frame deletions in the dystrophin gene. Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) converts out-of-frame to in-frame mutations, producing partially functional dystrophin. Four single-exon skipping PMOs are approved for DMD but treat only 8 to 14% of patients each, and some exhibit poor efficacy. Alternatively, exons 45 to 55 skipping could treat 40 to 47% of all patients and is associated with improved clinical outcomes. Here, we report the development of peptide-conjugated PMOs for exons 45 to 55 skipping. Experiments with immortalized patient myotubes revealed that exons 45 to 55 could be skipped by targeting as few as five exons. We also found that conjugating DG9, a cell-penetrating peptide, to PMOs improved single-exon 51 skipping, dystrophin restoration, and muscle function in hDMDdel52;mdx mice. Local administration of a minimized exons 45 to 55-skipping DG9-PMO mixture restored dystrophin production. This study provides proof of concept toward the development of a more economical and effective exons 45 to 55-skipping DMD therapy.

Natural History of a Mouse Model Overexpressing the Dp71 Dystrophin Isoform.

Dystrophin is a 427 kDa protein that stabilizes muscle cell membranes through interactions with the cytoskeleton and various membrane-associated proteins. Loss of dystrophin as in Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle weakness and cardiac dysfunction. Multiple promoters along the dystrophin gene (DMD) give rise to a number of shorter isoforms. Of interest is Dp71, a 71 kDa isoform implicated in DMD pathology by various animal and patient studies. Strong evidence supporting such a role for Dp71, however, is lacking. Here, we use del52;WT mice to understand how Dp71 overexpression affects skeletal and cardiac muscle phenotypes. Apart from the mouse Dmd gene, del52;WT mice are heterozygous for a full-length, exon 52-deleted human DMD transgene expected to only permit Dp71 expression in muscle. Thus, del52;WT mice overexpress Dp71 through both the human and murine dystrophin genes. We observed elevated Dp71 protein in del52;WT mice, significantly higher than wild-type in the heart but not the tibialis anterior. Moreover, del52;WT mice had generally normal skeletal muscle but impaired cardiac function, exhibiting significant systolic dysfunction as early as 3 months. No histological abnormalities were found in the tibialis anterior and heart. Our results suggest that Dp71 overexpression may have more detrimental effects on the heart than on skeletal muscles, providing insight into the role of Dp71 in DMD pathogenesis.

Identification and expression profiling of proline metabolizing genes in Arabidopsis thaliana and Oryza sativa to reveal their stress-specific transcript alteration.

The amino acid, proline, is utilized by different organisms to offset cellular imbalances caused by environmental stresses. The wide use of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. A comprehensive analysis of the transcript abundance of proline metabolizing genes is fundamental for the assessment of function and regulation of each gene. Using available microarray data and quantitative real-time RT-PCR, the expression profiles of gene encoding key proline biosynthesis and degradation enzymes i.e., OAT, P5CS, P5CR and PDH were examined. Interestingly, validation of candidate genes in rice using in-silico data provided strong evidence for their involvement in stress response. Note that, OsOAT, OsP5CS1, OsP5CS2, OsP5CR showed similar expression pattern in quantitative real-time RT-PCR results as compared to microarray data. However, OsPDH showed a different expression pattern which may be due to the genotypic variation. Furthermore, a biochemical assay measuring proline content gave us a proper indication of the accumulation of proline under stressed conditions. Identification of key proline metabolizing genes from rice and Arabidopsis provides insights on the molecular regulation of proline homeostasis, to initiate metabolic engineering to develop stress-resilient plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01023-0.

eSkip-Finder: a machine learning-based web application and database to identify the optimal sequences of antisense oligonucleotides for exon skipping.

Exon skipping using antisense oligonucleotides (ASOs) has recently proven to be a powerful tool for mRNA splicing modulation. Several exon-skipping ASOs have been approved to treat genetic diseases worldwide. However, a significant challenge is the difficulty in selecting an optimal sequence for exon skipping. The efficacy of ASOs is often unpredictable, because of the numerous factors involved in exon skipping. To address this gap, we have developed a computational method using machine-learning algorithms that factors in many parameters as well as experimental data to design highly effective ASOs for exon skipping. eSkip-Finder (https://eskip-finder.org) is the first web-based resource for helping researchers identify effective exon skipping ASOs. eSkip-Finder features two sections: (i) a predictor of the exon skipping efficacy of novel ASOs and (ii) a database of exon skipping ASOs. The predictor facilitates rapid analysis of a given set of exon/intron sequences and ASO lengths to identify effective ASOs for exon skipping based on a machine learning model trained by experimental data. We confirmed that predictions correlated well with in vitro skipping efficacy of sequences that were not included in the training data. The database enables users to search for ASOs using queries such as gene name, species, and exon number.

OdoBD: An online database for the dragonflies and damselflies of Bangladesh.

Combining scientific data over a long-time period is necessary for generating large-scale datasets, which are an essential component of comparative analysis for understanding evolutionary processes. Furthermore, monitoring temporal and spatial distributions of animals at a global and regional scale is essential for studying climate change driven extinction risks. Regional and global datasets focusing on different animal groups are on the rise to meet such challenges. Although being one of the earliest and best-known insect groups, the data on Odonata remains rudimentary and dispersed, especially in the South Asian region. Bangladesh, being located within a biodiversity hotspot, possesses a large number of odonate species and many of them are endemic to the South Asian region. We have developed an online database for the Odonata of Bangladesh by compiling and digitizing data from our last four years of field studies, from previously published research articles and field guides, and also by collecting data from citizen scientists. The Odonata of Bangladesh database (accessible at http://www.odobd.org) contains phenotypic, genotypic, photographic, taxonomic, biogeographic and faunistic data of the Odonata of Bangladesh. The database will be a valuable resource for understanding diversity, distributions, extinction risks and conservation planning of the Odonata of Bangladesh. Finally, phenotypic, spatial and temporal data of Odonata of Bangladesh datasets can be integrated with other regional datasets for analyzing macroevolutionary trends and to monitor the effect of climate change on odonates.

Molecular evolution of SUN-domain containing proteins in diverse plant species and their expression profiling in response to developmental and perturbation stimuli.

SUN (Sad1/UNC-84) domain-containing proteins are highly conserved throughout evolution. They are localized to the inner membrane of the nuclear envelope and are involved in nuclear migration and nucleoskeleton formation. In the present study, a genome-wide investigation was performed in three dicotyledonous (Arabidopsis thaliana, Glycine max and Medicago truncatula) and three monocotyledonous (Oryza sativa, Zea mays and Sorghum bicolor) plants. A total of 56 SUN proteins encoded by 30 genes were identified. Based on their length, transmembrane topology, conserved domains and phylogenetic relationships, they could be divided into two previously defined groups- Cter-SUN and mid-SUN proteins. Expression of these genes was analyzed in different developmental stages, tissues and various unfavorable conditions such as salinity, drought, and hormonal treatment. Analyses indicated that the expression of SUN1/2 transcripts are ubiquitous; that of SUN3/4 are development/tissue regulated, and SUN5 are inflorescence stage-specific. This study provides an initial framework for the characterization and functional validation of the plant SUN family.