Optimization of ACE-tRNAs function in translation for suppression of nonsense mutations.

Nonsense suppressor transfer RNAs (tRNAs) or AntiCodon-Edited tRNAs (ACE-tRNAs) have long been envisioned as a therapeutic approach to overcome genetic diseases resulting from the introduction of premature termination codons (PTCs). The ACE-tRNA approach for the rescue of PTCs has been hampered by ineffective delivery through available modalities for gene therapy. Here we have screened a series of ACE-tRNA expression cassette sequence libraries containing >1800 members in an effort to optimize ACE-tRNA function and provide a roadmap for optimization in the future.

Mechanism-based approach in designing patient-specific combination therapies for nonsense mutation diseases.

Premature termination codon (PTC) diseases, arising as a consequence of nonsense mutations in a patient's DNA, account for approximately 12% of all human disease mutations. Currently there are no FDA approved treatments for increasing PTC readthrough in nonsense mutation diseases, although one translational readthrough inducing drug, ataluren, has had conditional approval for treatment of Duchenne muscular dystrophy in Europe and elsewhere for 10 years. Ataluren displays consistent low toxicity in clinical trials for treatment of several different PTC diseases, but its therapeutic effects on such diseases are inconsistent.

Gz Enhanced Signal Transduction assaY (GESTY) for GPCR deorphanization.

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to G family members, however current assays to detect their activation are limited, hindering ligand identification efforts.

Verapamil mitigates chloride and calcium bi-channelopathy in a myotonic dystrophy mouse model.

Myotonic dystrophy type 1 (DM1) involves misregulated alternative splicing for specific genes. We used exon or nucleotide deletion to mimic altered splicing of genes central to muscle excitation-contraction coupling in mice. Mice with forced skipping of exon 29 in the CaV1.

Photomodulation of the ASIC1a acidic pocket destabilizes the open state.

Acid-sensing ion channels (ASICs) are important players in detecting extracellular acidification throughout the brain and body. ASICs have large extracellular domains containing two regions replete with acidic residues: the acidic pocket, and the palm domain. In the resting state, the acidic pocket is in an expanded conformation but collapses in low pH conditions as the acidic side chains are neutralized.

Combinatorial chloride and calcium channelopathy in myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) involves misregulated alternative splicing for specific genes. We used exon or nucleotide deletion to mimic altered splicing of genes central to muscle excitation-contraction coupling processes in mice. Mice with forced-skipping of exon 29 in Ca1.

Gene, RNA, and ASO-based therapeutic approaches in Cystic Fibrosis.

Most people with Cystic Fibrosis (PwCF) harbor Cystic Fibrosis Transmembrane Conductance (CFTR) mutations that respond to highly effective CFTR modulators (HEM); however, a small fraction of non-responsive variants will require alternative approaches for treatment. Furthermore, the long-term goal to develop a cure for CF will require novel therapeutic strategies. Nucleic acid-based approaches offer the potential to address all CF-causing mutations and possibly a cure for all PwCF.

Efficient suppression of endogenous CFTR nonsense mutations using anticodon-engineered transfer RNAs.

Nonsense mutations or premature termination codons (PTCs) comprise ∼11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases; however, their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells.

Therapeutic promise of engineered nonsense suppressor tRNAs.

Nonsense mutations change an amino acid codon to a premature termination codon (PTC) generally through a single-nucleotide substitution. The generation of a PTC results in a defective truncated protein and often in severe forms of disease. Because of the exceedingly high prevalence of nonsense-associated diseases and a unifying mechanism, there has been a concerted effort to identify PTC therapeutics.

β11-12 linker isomerization governs acid-sensing ion channel desensitization and recovery.

Acid-sensing ion channels (ASICs) are neuronal sodium-selective channels activated by reductions in extracellular pH. Structures of the three presumptive functional states, high-pH resting, low-pH desensitized, and toxin-stabilized open, have all been solved for chicken ASIC1. These structures, along with prior functional data, suggest that the isomerization or flipping of the β11-12 linker in the extracellular, ligand-binding domain is an integral component of the desensitization process.

Engineered transfer RNAs for suppression of premature termination codons.

Premature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. Here we present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid.

Orthogonality of Pyrrolysine tRNA in the Xenopus oocyte.

Chemical aminoacylation of orthogonal tRNA allows for the genetic encoding of a wide range of synthetic amino acids without the need to evolve specific aminoacyl-tRNA synthetases. This method, when paired with protein expression in the Xenopus laevis oocyte expression system, can extract atomic scale functional data from a protein structure to advance the study of membrane proteins. The utility of the method depends on the orthogonality of the tRNA species used to deliver the amino acid.

Cross-kingdom auxiliary subunit modulation of a voltage-gated sodium channel.

Voltage-gated, sodium ion-selective channels (Na) generate electrical signals contributing to the upstroke of the action potential in animals. Nas are also found in bacteria and are members of a larger family of tetrameric voltage-gated channels that includes Cas, Ks, and Nas. Prokaryotic Nas likely emerged from a homotetrameric Ca-selective voltage-gated progenerator, and later developed Na selectivity independently.

Cellular encoding of Cy dyes for single-molecule imaging.

A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins.

Atomic mutagenesis in ion channels with engineered stoichiometry.

C-type inactivation of potassium channels fine-tunes the electrical signaling in excitable cells through an internal timing mechanism that is mediated by a hydrogen bond network in the channels' selectively filter. Previously, we used nonsense suppression to highlight the role of the conserved Trp434-Asp447 indole hydrogen bond in Shaker potassium channels with a non-hydrogen bonding homologue of tryptophan, Ind (Pless et al., 2013).

Allele-specific gene silencing in two mouse models of autosomal dominant skeletal myopathy.

We explored the potential of mutant allele-specific gene silencing (ASGS) in providing therapeutic benefit in two established mouse models of the autosomal dominantly-inherited muscle disorders, Malignant Hyperthermia (MH) and Central Core Disease (CCD). Candidate ASGS siRNAs were designed and validated for efficacy and specificity on ryanodine receptor (RyR1) cDNA mini-constructs expressed in HEK293 cells using RT-PCR- and confocal microscopy-based assays. In vivo delivery of the most efficacious identified siRNAs into flexor digitorum brevis (FDB) muscles was achieved by injection/electroporation of footpads of 4-6 month old heterozygous Ryr1(Y524S/+) (YS/+) and Ryr1(I4895T/+) (IT/+) knock-in mice, established mouse models of MH with cores and CCD, respectively.

Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy.

Dystroglycan is a transmembrane glycoprotein that links the extracellular basement membrane to cytoplasmic dystrophin. Disruption of the extensive carbohydrate structure normally present on α-dystroglycan causes an array of congenital and limb girdle muscular dystrophies known as dystroglycanopathies. The essential role of dystroglycan in development has hampered elucidation of the mechanisms underlying dystroglycanopathies.

Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle.

Skeletal muscle fibers exhibit a high resting chloride conductance primarily determined by ClC-1 chloride channels that stabilize the resting membrane potential during repetitive stimulation. Although the importance of ClC-1 channel activity in maintaining normal muscle excitability is well appreciated, the subcellular location of this conductance remains highly controversial. Using a three-pronged multidisciplinary approach, we determined the location of functional ClC-1 channels in adult mouse skeletal muscle.

Genetic ablation of complement C3 attenuates muscle pathology in dysferlin-deficient mice.

Mutations in the dysferlin gene underlie a group of autosomal recessive muscle-wasting disorders denoted as dysferlinopathies. Dysferlin has been shown to play roles in muscle membrane repair and muscle regeneration, both of which require vesicle-membrane fusion. However, the mechanism by which muscle becomes dystrophic in these disorders remains poorly understood.

Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA.

Genomic expansions of simple tandem repeats can give rise to toxic RNAs that contain expanded repeats. In myotonic dystrophy, the expression of expanded CUG repeats (CUGexp) causes abnormal regulation of alternative splicing and neuromuscular dysfunction. We used a transgenic mouse model to show that derangements of myotonic dystrophy are reversed by a morpholino antisense oligonucleotide, CAG25, that binds to CUGexp RNA and blocks its interaction with muscleblind-like 1 (MBNL1), a CUGexp-binding protein.

Alternative splicing of RyR1 alters the efficacy of skeletal EC coupling.

Alternative splicing of ASI residues (Ala(3481)-Gln(3485)) in the skeletal muscle ryanodine receptor (RyR1) is developmentally regulated: the residues are present in adult ASI(+)RyR1, but absent in the juvenile ASI(-)RyR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RyR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr(3471)-Gly(3500)) in the splice variants.

Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy.

In myotonic dystrophy (dystrophia myotonica [DM]), an increase in the excitability of skeletal muscle leads to repetitive action potentials, stiffness, and delayed relaxation. This constellation of features, collectively known as myotonia, is associated with abnormal alternative splicing of the muscle-specific chloride channel (ClC-1) and reduced conductance of chloride ions in the sarcolemma. However, the mechanistic basis of the chloride channelopathy and its relationship to the development of myotonia are uncertain.

Muscle chloride channel dysfunction in two mouse models of myotonic dystrophy.

Muscle degeneration and myotonia are clinical hallmarks of myotonic dystrophy type 1 (DM1), a multisystemic disorder caused by a CTG repeat expansion in the 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Transgenic mice engineered to express mRNA with expanded (CUG)(250) repeats (HSA(LR) mice) exhibit prominent myotonia and altered splicing of muscle chloride channel gene (Clcn1) transcripts. We used whole-cell patch clamp recordings and nonstationary noise analysis to compare and biophysically characterize the magnitude, kinetics, voltage dependence, and single channel properties of the skeletal muscle chloride channel (ClC-1) in individual flexor digitorum brevis (FDB) muscle fibers isolated from 1-3-wk-old wild-type and HSA(LR) mice.

Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1.

Transmembrane chloride ion conductance in skeletal muscle increases during early postnatal development. A transgenic mouse model of myotonic dystrophy type 1 (DM1) displays decreased sarcolemmal chloride conductance. Both effects result from modulation of chloride channel 1 (CLCN1) expression, but the respective contributions of transcriptional vs.