MicroRNA therapeutics: a new niche for antisense nucleic acids

MicroRNA molecules (miRNAs) are naturally occurring triggers of the RNA-interference pathway. The first identified miRNA, lin-4, was discovered in Caenorhabditis elegans >20 years ago. What began as a curiosity in this model organism has expanded into almost every area of biology; there are now 326 confirmed miRNA genes in humans and the total is predicted to reach 1000. Each miRNA has the potential to regulate hundreds of mRNAs; therefore, there are likely to be few biological pathways not impacted by miRNA regulation. Recent evidence has suggested that miRNAs might be viable therapeutic targets for a wide range of diseases, including cancer. A recent article by Stoffel and colleagues has demonstrated remarkably effective inhibition of miRNAs in vivo, thus providing an entry point into the promising new arena of miRNA therapeutics.     

Discovery and development of respirable antisense therapeutics for asthma

Respirable antisense oligonucleotides (RASONs) represent a novel class of respiratory therapeutic molecules with the potential to specifically address the challenges posed by the successes of the Human Genome Program, namely, the need to rapidly identify the critical pulmonary disease-relevant drugable targets from the vast pool of 30,000-40,000 human genes and to discover and develop drugs that specifically attack these targets. We have shown that EPI-2010, a RASON targeting the adenosine A1 receptor, a G-protein coupled receptor that has been implicated in the regulation of three major determinants of asthma, can be delivered directly to the target disease tissue as an aerosol formulation. In vivo efficacy, absorption, distribution, metabolism, and excretion (ADME), and safety studies of inhaled EPI-2010 employing animal models of human asthma suggest that the RASON approach enables the specific delivery of efficacious, safe, and long-acting doses of phosphorothioate oligonucleotides to the respiratory tract. Moreover, these data indicate that RASONs truly have the potential to address the respiratory drug discovery bottleneck of the postgenomic era, that is, the ability to rapidly validate disease targets and develop pulmonary disease therapeutics for these validated targets.     

Polypyrimidine tract-binding protein 2 binds to selective, intronic messenger RNA and microRNA targets in the mouse testis

Here we use an in vivo cross-linking and immunoprecipitation procedure to detect RNA targets of the multifunctional RNA-binding protein polypyrimidine tract-binding protein (PTBP) 2 in mouse testis. Eleven known mRNAs, including Ptbp2 mRNA, 28 RNAs matching intron sequences, and 12 small RNAs and repeat sequences are identified. The specificity of interaction between PTBP2 and its target RNAs was confirmed using RNA interference with mouse N2A cells. Reduction of PTBP2 levels led to decreases in 7 of 10 of the mRNAs, to the repression of alternative splicing of introns, and to reductions in specific miRNAs.     

RNA interference trigger variants: getting the most out of RNA for RNA interference-based therapeutics

The manifestation of RNA interference (RNAi)-based therapeutics lies in safe and successful delivery of small interfering RNAs (siRNAs), the molecular entity that triggers and guides sequence-specific degradation of target mRNAs. Optimizing the chemistry and structure of siRNAs to achieve maximum efficacy is an important parameter in the development of siRNA therapeutics. The RNAi protein machinery can tolerate a variety of non-canonical modifications made to siRNAs, each of which imparts advantageous properties. Here, we review these modifications to siRNAs in pre-clinical and clinical studies.     

Another role for RNA: a messenger across generations

There has been a recent resurgence of interest in the notion that DNA is not the sole determinant of our inherited phenotype. The strongest evidence for transgenerational epigenetic inheritance has come from studies of paramutation in plants. But few examples have been reported in other species, and the molecular basis for the process has been unclear. A recent paper by Rassoulzadegan and colleagues provides clear evidence of paramutation in mice and strong evidence that RNA is involved.     

Homology modeling of human methylmalonyl-CoA mutase: a structural basis for point mutations causing methylmalonic aciduria.

Point mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)-dependent methylmalonyl-CoA mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic alpha-subunit of the heterodimeric methylmalonyl-CoA mutase from the bacterium Propionibacterium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three-dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X-ray crystal structure of the P. shermanii methylmalonyl-CoA mutase. Eight mutations are found in the C-terminal B12-binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin-like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent-exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N-terminal region of the mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl-CoA esters gain access to the active site (W105R). The extreme N-terminus of methylmalonyl-CoA mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.     

Crosslinking transfer RNA and messenger RNA at the ribosomal decoding region: identification of the site of reaction on the messenger RNA.

Wybutine (Ywye), situated next to the 3'-side of the anticodon of tRNAPhe from Saccharomyces cerevisiae, can be photo-crosslinked to mRNA when bound to Escherichia coli ribosomes. Crosslinking can be obtained with poly(U) as well as with oligonucleotides such as pAUGUUU or p(U)6. In order to identify the site of reaction on the mRNA, 5'-[32P]-labelled pAUGUUU was crosslinked by irradiation at 320 nm with Phe-tRNAPhe from yeast bound to the acceptor-site. The photoproduct was subsequently digested with P1-nuclease and analyzed by electrophoresis followed by homochromatography in the second dimension. As a result of the photoreaction the wybutine was found to be crosslinked to the U at the 5'-position of the corresponding UUU-codon.     

Masked antisense: a molecular configuration for discriminating similar RNA targets

Antisense technology has great potential for the control of RNA expression, but there remain few successful applications of the technology. Expressed antisense RNA can effectively down-regulate expression of a gene over long periods, but cannot differentiate partly identical sequences, such as the mRNA of fusion genes or those with point mutants. We have designed a structured form of expressed antisense, which can discriminate between highly similar mRNA molecules. These ‘masked’ antisense RNAs have most of the antisense sequence sequestered within duplex elements, leaving a short single-stranded region to initiate binding to target RNA. After contacting the correct target, the structured RNA can unravel, releasing the masked antisense region to form a stable duplex with the mRNA. We demonstrate that suitable masked antisense RNA can discriminate between the two forms of BCR–ABL mRNA that result from the Philadelphia chromosomal translocations, as well as discriminating the normal BCR and ABL mRNA.     

Cloning and regulation of messenger RNA for mouse apolipoprotein E

A cDNA clone for mouse apolipoprotein E has been identified from a mouse liver cDNA library by a combination of differential colony hybridization and hybrid selection-translation. The identity of the clone was unambiguously established by partial sequencing and comparison with human apolipoprotein E nucleotide and amino acid sequences. In conjunction with an in vitro translation assay for apolipoprotein E, the clone has been used to examine the relative levels of apolipoprotein E mRNA in various tissues of the mouse and the regulation of apolipoprotein E synthesis in response to a diet rich in saturated fat and cholesterol. In the tissues examined, the clone was found to hybridize to a polyadenylated RNA species of approximately 1400 nucleotides. Of the tissues involved in lipoprotein synthesis, liver is very rich (about 1% of total) in apolipoprotein E mRNA while intestine contains only trace amounts. Appreciable levels of active apolipoprotein E mRNA (up to 10% of that in liver) are also detected in peripheral tissues not associated with lipoprotein synthesis, including lung, kidney, spleen, and heart. Thus, extrahepatic apolipoprotein E synthesis may contribute significantly to the levels present in plasma, and a possible function in "reverse cholesterol transport" is considered. When mice were placed on a high lipid diet there was no discernible change in the level of apolipoprotein E mRNA in liver or intestine, although the level of the circulating protein increased about 3-fold. We conclude that in mice the effect of diet on apolipoprotein E levels in blood does not result from induction of mRNA in these tissues.