Selective Delivery to Cardiac Muscle Cells Using Cell-Specific Aptamers.

In vivo SELEX is an advanced adaptation of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that allows the development of aptamers capable of recognizing targets directly within their natural microenvironment. While this methodology ensures a higher translation potential for the selected aptamer, it does not select for aptamers that recognize specific cell types within a tissue. Such aptamers could potentially improve the development of drugs for several diseases, including neuromuscular disorders, by targeting solely the proteins involved in their pathogenesis. Here, we describe our attempt to utilize in vivo SELEX with a modification in the methodology that drives the selection of intravenously injected aptamers towards a specific cell type of interest. Our data suggest that the incorporation of a cell enrichment step can direct the in vivo localization of RNA aptamers into cardiomyocytes, the cardiac muscle cells, more readily over other cardiac cells. Given the crucial role of cardiomyocytes in the disease pathology in DMD cardiomyopathy and therapy, these aptamers hold great potential as drug delivery vehicles with cardiomyocyte selectivity.

Intramuscular Evaluation of Chimeric Locked Nucleic Acid/2'OMethyl-Modified Antisense Oligonucleotides for Targeted Exon 23 Skipping in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a fatal disorder characterised by progressive muscle wasting. It is caused by mutations in the dystrophin gene, which disrupt the open reading frame leading to the loss of functional dystrophin protein in muscle fibres. Antisense oligonucleotide (AON)-mediated skipping of the mutated exon, which allows production of a truncated but partially functional dystrophin protein, has been at the forefront of DMD therapeutic research for over two decades. Nonetheless, novel nucleic acid modifications and AON designs are continuously being developed to improve the clinical benefit profile of current drugs in the DMD pipeline. We herein designed a series of 15mer and 20mer AONs, consisting of 2'O-Methyl (2'OMe)- and locked nucleic acid (LNA)-modified nucleotides in different percentage compositions, and assessed their efficiency in inducing exon 23 skipping and dystrophin restoration in locally injected muscles of mdx mice. We demonstrate that LNA/2'OMe AONs with a 30% LNA composition were significantly more potent in inducing exon skipping and dystrophin restoration in treated mdx muscles, compared to a previously tested 2'OMe AON and LNA/2'OMe chimeras with lower or higher LNA compositions. These results underscore the therapeutic potential of LNA/2'OMe AONs, paving the way for further experimentation to evaluate their benefit-toxicity profile following systemic delivery.

Systemic Evaluation of Chimeric LNA/2'-O-Methyl Steric Blockers for Myotonic Dystrophy Type 1 Therapy.

Myotonic dystrophy type 1 (DM1) is a dominantly inherited, multisystemic disorder characterized clinically by delayed muscle relaxation and weakness. The disease is caused by a CTG repeat expansion in the 3' untranslated region (3' UTR) of the DMPK gene, which leads to the expression of a toxic gain-of-function mRNA. The expanded CUG repeat mRNA sequesters the MBNL1 splicing regulator in nuclear-retained foci structures, resulting in loss of protein function and disruption of alternative splicing homeostasis. In this study, we used CAG repeat antisense oligonucleotides (ASOs), composed of locked nucleic acid (LNA)- and 2'-O-methyl (2'OMe)-modified bases in a chimeric design, to alleviate CUGexpanded-mediated toxicity. Chimeric 14-18mer LNA/2'OMe oligonucleotides, exhibiting an LNA incorporation of ∼33%, significantly ameliorated the misregulated alternative splicing of Mbnl1-dependent exons in primary DM1 mouse myoblasts and tibialis anterior muscles of DM1 mice. Subcutaneous delivery of 14mer and 18mer LNA/2'OMe chimeras in DM1 mice resulted in high levels of accumulation in all tested skeletal muscles, as well as in the diaphragm and heart tissue. Despite the efficient delivery, chimeric LNA/2'OMe oligonucleotides were not able, even at a high-dosage regimen (400 mg/kg/week), to correct the misregulated splicing of Serca1 exon 22 in skeletal muscles. Nevertheless, oligonucleotide doses were well-tolerated as determined by histological and plasma biochemistry analyses. Our results provide proof of concept that inhibition of MBNL1 sequestration by systemic delivery of a steric-blocking ASO is extremely challenging, considering the large number of target sites that need to be occupied per RNA molecule. Although not suitable for DM1 therapy, chimeric LNA/2'OMe oligonucleotides could prove to be highly beneficial for other diseases, such as Duchenne muscular dystrophy, that require inhibition of a single target site per RNA molecule.

Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers.

Oligonucleotide gene therapy has shown great promise for the treatment of muscular dystrophies. Nevertheless, the selective delivery to affected muscles has shown to be challenging because of their high representation in the body and the high complexity of their cell membranes. Current trials show loss of therapeutic molecules to non-target tissues leading to lower target efficacy. Therefore, strategies that increase uptake efficiency would be particularly compelling. To address this need, we applied a cell-internalization SELEX (Systematic Evolution of Ligands by Exponential Enrichment) approach and identified a skeletal muscle-specific RNA aptamer. A01B RNA aptamer preferentially internalizes in skeletal muscle cells and exhibits decreased affinity for off-target cells. Moreover, this in vitro selected aptamer retained its functionality in vivo, suggesting a potential new approach for targeting skeletal muscles. Ultimately, this will aid in the development of targeted oligonucleotide therapies against muscular dystrophies.

miR-186 inhibits muscle cell differentiation through myogenin regulation.

The complex process of skeletal muscle differentiation is organized by the myogenic regulatory factors (MRFs), Myf5, MyoD, Myf6, and myogenin, where myogenin plays a critical role in the regulation of the final stage of muscle differentiation. In an effort to investigate the role microRNAs (miRNAs) play in regulating myogenin, a bioinformatics approach was used and six miRNAs (miR-182, miR-186, miR-135, miR-491, miR-329, and miR-96) were predicted to bind the myogenin 3'-untranslated region (UTR). However, luciferase assays showed only miR-186 inhibited translation and 3'-UTR mutagenesis analysis confirmed this interaction was specific. Interestingly, the expression of miR-186 mirrored that of its host gene, ZRANB2, during development. Functional studies demonstrated that miR-186 overexpression inhibited the differentiation of C2C12 and primary muscle cells. Our findings therefore identify miR-186 as a novel regulator of myogenic differentiation.

Twist reverses muscle cell differentiation through transcriptional down-regulation of myogenin.

Some higher vertebrates can display unique muscle regenerative abilities through dedifferentiation. Research evidence suggests that induced dedifferentiation can be achieved in mammalian cells. TWIST is a bHLH (basic helix-loop-helix) transcription factor that is expressed during embryonic development and plays critical roles in diverse developmental systems including myogenesis. Several experiments demonstrated its role in inhibition of muscle cell differentiation. We have previously shown that overexpression of TWIST can reverse muscle cell differentiation in the presence of growth factors. Here we show that TWIST reverses muscle cell differentiation through binding and down-regulation of myogenin. Moreover, it can reverse cellular morphology in the absence of growth factors.

Identification and characterization of a novel X-linked AVPR2 mutation causing partial nephrogenic diabetes insipidus: a case report and review of the literature.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease characterized by a malfunctioning renal response to the antidiuretic hormone arginine vasopressin (AVP) due to mutations in the AVPR2 gene. A limited number of mutations in the AVPR2 gene resulting in partial phenotype have been described so far. In this mini-review the retrospective analysis of 13 known AVPR2 mutations that have been previously shown in vitro to partially abolish AVPR2 function is described, along with a novel mutation diagnosed in a kindred with partial NDI. In the present study, a 14 year old male and his 73 year old maternal grandfather were diagnosed with partial NDI based on the clinical phenotype, the water deprivation test and the inadequate response to 1-desamino-8-d-arginine vasopressin (DDAVP) administration. Sequencing analysis of the AVPR2 gene revealed the novel missense mutation p.N317S (g.1417A > G) in both patients. This mutation was re-created by site directed mutagenesis in an AVPR2 cDNA expression vector and was functionally characterized, in terms of arginine vasopressin (AVP) and DDAVP response. AVPR2 activity of the p.N317S mutant receptor after the AVP and DDAVP administration, as assessed by cAMP production was reduced and impaired when compared to cells that expressed the wild type AVPR2 gene. In conclusion, the affected members of this family have X-linked NDI with partial resistance to AVP, due to a missense mutation in the AVPR2 gene.

Down-regulation of myogenin can reverse terminal muscle cell differentiation.

Certain higher vertebrates developed the ability to reverse muscle cell differentiation (dedifferentiation) as an additional mechanism to regenerate muscle. Mammals, on the other hand, show limited ability to reverse muscle cell differentiation. Myogenic Regulatory Factors (MRFs), MyoD, myogenin, Myf5 and Myf6 are basic-helix-loop-helix (bHLH) transcription factors essential towards the regulation of myogenesis.Our current interest is to investigate whether down-regulation of MRFs in terminally differentiated mouse myotubes can induce reversal of muscle cell differentiation. Results from this work showed that reduction of myogenin levels in terminally differentiated mouse myotubes can reverse their differentiation state. Down-regulation of myogenin in terminally differentiated mouse myotubes induces cellular cleavage into mononucleated cells and cell cycle re-entry, as shown by re-initiation of DNA synthesis and increased cyclin D1 and cyclin E2 levels. Finally, we provide evidence that down-regulation of myogenin causes cell cycle re-entry (via down-regulation of MyoD) and cellularisation through separate pathways. These data reveal the important role of myogenin in maintaining terminal muscle cell differentiation and point to a novel mechanism by which muscle cells could be re-activated through its down-regulation.

Endogenous TWIST expression and differentiation are opposite during human muscle development.

TWIST is a transcription factor expressed during early embryonic development. In this study we investigate the expression of TWIST during human muscle development. Human TWIST was found to be endogenously expressed in human fetal myoblasts, and its expression decreased during late stages of development. Myoblasts showed an increasing capacity to differentiate in vitro during development. This inversely proportional relation between TWIST and differentiation capacity of myoblasts suggests that TWIST is involved in the regulation of muscle development.

Expression of miR-1, miR-133a, miR-133b and miR-206 increases during development of human skeletal muscle.

BACKGROUND: MicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally regulate gene expression and have been shown to play an important role during development. miR-1, miR-133a, miR-133b and miR-206 are expressed in muscle tissue and induced during muscle cell differentiation, a process that directs myoblasts to differentiate into mature myotubes, which are organized into myofibers. Although miR-1, miR-133a, miR-133b and miR-206 are well-studied in muscle, there is no information about their expression and function during human development. The purpose of this study was to determine the profile of these miRNAs in muscle cells isolated from different stages of human development. RESULTS: We examined the levels of miR-1, miR-133a, miR-133b and miR-206 during the development of human foetus. All four miRNA levels were found increased during late stages of human foetal muscle development. Increases in the expression levels of these miRNAs were proportional to the capacity of myoblasts to form myotubes. Changes in miRNA levels during human foetal development were accompanied by endogenous alterations in their known targets and also in their inducer, MyoD. Ectopic MyoD expression caused an induction of muscle cell differentiation in vitro, accompanied by an increase in the levels of miR-1, miR-133a, miR-133b and miR-206. CONCLUSIONS: This study provides data about the profile of four miRNAs in human muscle cells isolated during different stages of foetal development. These results may shed light on the differentiation of muscle cells and regulation of muscle formation through miRNAs, during the development of human foetus.

Hydrophilic cationic star homopolymers based on a novel diethanol-N-methylamine dimethacrylate cross-linker for siRNA transfection: synthesis, characterization, and evaluation.

Four cationic hydrophilic star homopolymers based on the novel hydrophilic, positively ionizable cross-linker bis(methacryloyloxyethyl)methylamine (BMEMA) were synthesized using sequential group transfer polymerization (GTP) and were, subsequently, evaluated for their ability to deliver siRNA to mouse myoblast cells. The nominal degrees of polymerization (DP) of the arms were varied from 10 to 50. For the polymerizations, 2-(dimethylamino)ethyl methacrylate (DMAEMA) was employed as the hydrophilic, positively ionizable monomer. For comparison, four linear DMAEMA homopolymers were also synthesized, whose nominal DPs were the same as those of the arms of the stars. The numbers of arms of the star homopolymers were determined using gel permeation chromatography with static light scattering detection, and found to range from 7 to 19, whereas the hydrodynamic diameters of the star homopolymers in aqueous solution were measured using dynamic light scattering and found to increase with the arm DP from 13 to 26 nm. The presence of the hydrophilic BMEMA cross-linker enabled the solubility of all star homopolymers in pure water. The cloud points of the star homopolymers in aqueous solution increased with the arm DP from 23 to 29 °C, while the cloud points of the linear homopolymers were found to decrease with their DP, from 42 to 32 °C. The effective pK values of the DMAEMA units were in the range of 6.9 to 7.3 for the star homopolymers, whereas they ranged between 7.3 and 7.4 for the linear homopolymers. Subsequently, all star and linear homopolymers were evaluated for their ability to deliver siRNA to the C2C12 mouse myoblast cell line, expressing the reporter enhanced green fluorescent protein (EGFP). All star homopolymers and the largest linear homopolymer presented significant EGFP suppression, whereas the smaller linear homopolymers were much less efficient. For all star homopolymers and the largest linear homopolymer both the EGFP suppression and the cell toxicity increased with polymer loading. The siRNA-specific EGFP suppression, calculated by subtracting the effect of cell toxicity on EGFP suppression, slightly increased with star polymer loading for the two smaller stars, whereas it presented a shallow maximum and a decrease for the other two stars. Moreover, the siRNA-specific EGFP suppression also increased slightly with the DP of the arms of the DMAEMA star homopolymers. Overall, the EGFP suppression efficiencies with the present star homopolymers were at levels comparable to that of the commercially available transfection reagent Lipofectamine.

The application of ribozymes and DNAzymes in muscle and brain.

The discovery of catalytic nucleic acids (CNAs) has provided scientists with valuable tools for the identification of new therapies for several untreated diseases through down regulation or modulation of endogenous gene expression involved in these ailments. These CNAs aim either towards the elimination or repair of pathological gene expression. Ribozymes, a class of CNAs, can be mostly used to down-regulate (by RNA cleavage) or repair (by RNA trans-splicing) unwanted gene expression involved in disease. DNAzymes, derived by in vitro selection processes are also able to bind and cleave RNA targets and therefore down-regulate gene expression. The purpose of this review is to present and discuss several applications of ribozymes and DNAzymes in muscle and brain. There are several diseases which affect muscle and brain and catalytic nucleic acids have been used as tools to target specific cellular transcripts involved in these groups of diseases.

The effect of myotonic dystrophy transcript levels and location on muscle differentiation.

In myotonic dystrophy type I (DM1), nuclear retention of mutant DMPK transcripts compromises muscle cell differentiation. Although several reports have identified molecular defects in myogenesis, it remains still unclear how exactly the retention of the mutant transcripts induces this defect. We have recently created a novel cellular model in which the mutant DMPK 3' UTR transcripts were released to the cytoplasm of myoblasts by using the WPRE genetic element. As a result, muscle cell differentiation was repaired. In this paper, this cellular model was further exploited to investigate the effect of the levels and location of the mutant transcripts on muscle differentiation. Results show that the levels of these transcripts were proportional to the inhibition of both the initial fusion of myoblasts and the maturity of myotubes. Moreover, the cytoplasmic export of the mutant RNAs to the cytoplasm caused less inhibition only in the initial fusion of myoblasts.

Woodchuck post-transcriptional element induces nuclear export of myotonic dystrophy 3' untranslated region transcripts.

The woodchuck post-transcriptional regulatory element (WPRE) can naturally accumulate hepatitis transcripts in the cytoplasm, and has been recently exploited as an enhancer of transgene expression. The retention of mutant myotonic dystrophy protein kinase (DMPK) transcripts in the nucleus of myotonic dystrophy (DM) cells has an important pathogenic role in the disease, resulting in pleiotropic effects including delayed myoblast differentiation. In this study, we report the first use of WPRE as a tool to enhance nuclear export of an aberrantly retained messenger RNA. Stable cell lines expressing the normal and mutant DMPK 3' UTR (3' untranslated region) complementary DNA, with or without WPRE, were produced. It is noteworthy that WPRE stimulated extensive transport of mutant transcripts to the cytoplasm. This was associated with repair of the defective cellular MyoD levels and a subsequent increase in myoblast differentiation. These results provide the basis for a cellular model that can be exploited in DM and in the study of RNA transport mechanisms.