RNA Trans-Splicing Modulation via Antisense Molecule Interference.

The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense beta-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sense-strand globin mRNA.

pCGR2 and pCG1 belong to different subfamilies of the pCG1 family of Corynebacterium glutamicum plasmids. Nonetheless, they harbour homologous putative antisense RNA genes, crrI and cgrI, respectively. The genes in turn share identical positions complementary to the leader region of their respective repA (encoding plasmid replication initiator) genes. Determination of their precise transcriptional start- and end-points revealed the presence of short antisense RNA molecules (72 bp, CrrI; and 73 bp, CgrI). These short RNAs and their target mRNAs were predicted to form highly structured molecules comprising stem-loops with known U-turn motifs. Abolishing synthesis of CrrI and CgrI by promoter mutagenesis resulted in about sevenfold increase in plasmid copy number on top of an 11-fold (CrrI) and 32-fold (CgrI) increase in repA mRNA, suggesting that CrrI and CgrI negatively control plasmid replication. This control is accentuated by parB, a gene that encodes a small centromere-binding plasmid-partitioning protein, and is located upstream of repA. Simultaneous deactivation of CrrI and parB led to a drastic 87-fold increase in copy number of a pCGR2-derived shuttle vector. Moreover, the fact that changes in the structure of the terminal loops of CrrI and CgrI affected plasmid copy number buttressed the important role of the loop structure in formation of the initial interaction complexes between antisense RNAs and their target mRNAs. Similar antisense RNA control systems are likely to exist not only in the two C. glutamicum pCG1 subfamilies but also in related plasmids across Corynebacterium species.

Spinal Muscular Atrophy is a leading genetic cause of infantile death and occurs in approximately 1/6000 live births. SMA is caused by the loss of Survival Motor Neuron-1 (SMN1), however, all patients retain at least one copy of a nearly identical gene called SMN2. While SMN2 and SMN1 are comprised of identical coding sequences, the majority of SMN2 transcripts are alternatively spliced, encoding a truncated protein that is unstable and non-functional. Considerable effort has focused upon modulating the SMN2 alternative splicing event since this would produce more wildtype protein. Recently we reported the development of an optimized trans-splicing system that involved the co-expression of a SMN2 trans-splicing RNA and an anti-sense RNA that blocks a downstream splice site in SMN2 pre-mRNA. Here we demonstrate that in vivo delivery of the optimized trans-splicing vector increases an important SMN-dependent activity, snRNP assembly, in disease-relevant tissue in the SMA mouse model. A single injection of the vector into the intracerebral-ventricular space in SMA neonates also lessens the severity of the SMA phenotype in a severe SMA mouse model, extending survival ~70%. Collectively, these results provide the first in vivo demonstration that SMN2 trans-splicing leads to a lessening of the severity of the SMA phenotype and provide evidence for the power of this strategy for reprogramming genetic diseases at the pre-mRNA level.

The antisense RNA molecule is a unique DNA transcript consisting of 19-23 nucleotides, characterized by its complementary nature to mRNA. These antisense RNAs play a crucial role in regulating gene expression at various stages, including replication, transcription, and translation. Additionally, artificial antisense RNAs have demonstrated their ability to effectively modulate gene expression in host cells. Consequently, there has been a substantial increase in research dedicated to investigating the roles of antisense RNAs. These molecules have been found to be influential in various cellular processes, such as X-chromosome inactivation and imprinted silencing in healthy cells. However, it is important to recognize that in cancer cells; aberrantly expressed antisense RNAs can trigger the epigenetic silencing of tumor suppressor genes. Moreover, the presence of deletion-induced aberrant antisense RNAs can lead to the development of diseases through epigenetic silencing. One area of drug development worth mentioning is antisense oligonucleotides (ASOs), and a prime example of an oncogenic trans-acting long noncoding RNA (lncRNA) is HOTAIR (HOX transcript antisense RNA). NATs (noncoding antisense transcripts) are dysregulated in many cancers, and researchers are just beginning to unravel their roles as crucial regulators of cancer's hallmarks, as well as their potential for cancer therapy. In this review, we summarize the emerging roles and mechanisms of antisense RNA and explore their application in cancer therapy.

Moloney murine leukemia virua (MoMuLV)-derived retroviral vectors were engineered to express human immunodeficiency virus type 1 (HIV-1) packaging (psi) signal and Rev response element (RRE) sequences in either sense or antisense orientation. The RRE sequences were expressed under the control of the herpes simplex virus (HSV) thymidine kinase (tk) promoter fused to the HIV-1 trans-activation-responsive (TAR) element, while the psi signal sequences were expressed under control of the HSV tk promoter. Both RRE and psi signal sequences were expressed as part of the 3' untranslated region of the neomycin phosphotransferase (neo) mRNA. The constructs were used to transfect/infect packaging cell lines and the retroviral vector particles released were used to infect a human CD4+ lymphocyte-derived MT4 cell line. The stable MT4 transformants, harboring proviral vector DNA expressing one to two copies of HIV-1 RRE and psi signal in either antisense or sense orientation, were each tested for their susceptibility to HIV-1 infection. Compared to the results obtained with the control cells lacking any of the test DNA sequences, the rate of HIV-1 production remained unaltered in RRE1+ (sense RNA containing a single copy of RRE) RNA-containing cells, whereas it was delayed in cells expressing both RRE2+ (sense RNA containing two copies of RRE) and RRE1- (antisense RNA containing a single copy of RRE) RNA-expressing cells. In cells expressing HIV-1 psi signal, HIV-1 production remained unaltered in psi + RNA-expressing cells, whereas it was delayed by up to 30 days in psi - RNA-expressing cells.(ABSTRACT TRUNCATED AT 250 WORDS)

ABSTRACTThe transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules, followed by similar proportions of mRNA transcripts and noncoding RNAs. The two platforms detected similar proportions of uncharged tRNAs and 29 yet-undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNAArg, and the tRNAArg gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct nonoverlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range (a few hundred to thousands of bases) and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet-undescribed RNA molecules and an unexpected composition of the pools of tRNAs and sense and antisense RNAs.IMPORTANCE Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of rRNAs, enzymatic reverse transcription, and the fragmentation, size selection, and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the Nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that—if replicated in other species—will change our view of the bacterial RNA world. The discovery of sense-antisense pairs of transfer-messenger RNA (tmRNA), tRNAs, and mRNAs indicates a further and unknown level of gene regulation in bacteria.

Conventional wisdom holds that only one of the two strands in a microRNA (miRNA) precursor duplex is selected as the active guide strand. The complementary passenger strand is thought to be inactive. In triple negative breast cancer (TNBC), high levels of the miRNA guide strand called miR-17-5p inhibits ribosomal translation of tumor suppressor genes, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, we found that antisense DNA-LNA knockdown of miR-17-5p guide strand reduced PDCD4 and PTEN proteins by 1.8±0.3 fold in human MDA-MB-231 TNBC cells, instead of raising them. Due to high sequence homology between the antisense molecules and miR-17-3p passenger strand, we began seeking an answer to the contradiction by in silico analysis. We found 4 putative binding sites for miR-17-3p passenger strand in PDCD4 mRNA, and 6 in PTEN mRNA, but only one in either mRNA for the miR-17-5p guide strand. We investigated the possibility that gene targets of miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by miR-17-3p passenger strand. As a result, antisense knockdown of miR-17-3p raised PDCD4 mRNA by 25±2% and PTEN mRNA by 22±6%. Transfection of miR-17-5p or miR-17-3p RNA mimics into TNBC cells reduced PDCD4 and PTEN protein levels. From these results, we speculated that, similar to miR-17-5p guide strand, miR-17-3p passenger strand might regulate the translation of PDCD4 and PTEN mRNAs. Moreover, the antisense DNA-LNA against miR-17-5p guide strand mimicked miR-17-3p passenger strand, effectively raising the miR-17-3p concentration in TNBC cells. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand. To challenge our new hypothesis, luciferase assays using reporter constructs harboring miR-17-5p and/or miR-17-3p binding sites from the 3′UTR of PDCD4 and PTEN mRNAs are underway. No conflicts of interest. Supported by NIH CA148565 to E.W. Note: This abstract was not presented at the meeting. Citation Format: Yuan-Yuan Jin, Nicole L. Simone, Eric Wickstrom. Antisense agents and RNA mimics for miR-17-5p guide strand and miR-17-3p passenger strand differentiate the strength of guide and passenger strand targets in PDCD4 and PTEN mRNA 3′UTRs in MDA-MB-231 triple negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3963. doi:10.1158/1538-7445.AM2015-3963

The frequency of inherited malformations as well as genetic disorders in newborns account for around 3-5%. These frequency is much higher in early stages of pregnancy, because serious malformations and genetic disorders usually lead to spontaneous abortion. Prenatal diagnosis allowed identification of malformations and/or some genetic syndromes in fetuses during the first trimester of pregnancy. Thereafter, taking into account the severity of the disorders the decision should be taken in regard of subsequent course of the pregnancy taking into account a possibilities of treatment, parent's acceptation of a handicapped child but also, in some cases the possibility of termination of the pregnancy. In prenatal testing, both screening and diagnostic procedures are included. Screening procedures such as first and second trimester biochemical and/or ultrasound screening, first trimester combined ultrasound/biochemical screening and integrated screening should be widely offered to pregnant women. However, interpretation of screening results requires awareness of both sensitivity and predictive value of these procedures. In prenatal diagnosis ultrasound/MRI searching as well as genetic procedures are offered to pregnant women. A variety of approaches for genetic prenatal analyses are now available, including preimplantation diagnosis, chorion villi sampling, amniocentesis, fetal blood sampling as well as promising experimental procedures (e.g. fetal cell and DNA isolation from maternal blood). An incredible progress in genetic methods opened new possibilities for valuable genetic diagnosis. Although karyotyping is widely accepted as golden standard, the discussion is ongoing throughout Europe concerning shifting to new genetic techniques which allow obtaining rapid results in prenatal diagnosis of aneuploidy (e.g. RAPID-FISH, MLPA, quantitative PCR).

Anti-picornaviral antisense agents are part of a broader group of nucleic acid-based molecules developed for sequence-specific inhibition of translation and/or transcription of the target sequence through induced nuclease activity or physical hindrance. Three types of nucleic acid-based gene silencing molecules can be distinguished, including DNA-base antisense oligonucleotides (ASO), nucleic acid enzymes (ribozyme and DNAzyme) and double-stranded small interfering RNA (siRNA or microRNA). These antisense DNA and RNA molecules have been widely studied for gene functional studies and therapeutic purposes. In this review, we focus on drug development using ASO and siRNA strategies to inhibit picornavirus infections. The picornavirus genome organization and life cycle is described, followed by discussion of design considerations, chemical modifications and drug delivery approaches. Recent studies using antisense against picornavirus are reviewed. Finally, we compare the advantages and disadvantages of the antisense agents with those of other therapeutics, taking into consideration their limitations which need to be overcome to achieve the final goal of clinical application.

New paradigms for gene transfer: RNA trans-splicing and small interfering RNA as therapeutic strategies

The application of antisense oligodeoxynucleotides as tools for specific inhibition of gene expression was an extremely important step in the development of molecular medicine (reviewed in ref. 1, see also Chapters 3 and 11). From the first moment of experimental investigation of the power of these molecules it was obvious that, at some stage, this class of molecules would find its way to clinical application as therapeutic drugs (2). After several years of successful use in cell culture experiments, antisense molecules including synthetic oligos and expressed RNA are increasingly applied in animal experiments. Most of the studies carried out in cdl culture and animals so far have been aimed at the investigation of the consequences of the loss of a particular function. The antisense strategy has the advantage over the powerful method of gene knock-outs of allowing transient suppression of gene expression. Thus. it allows one to inactivate, for a short period of time, those functions which would be lethal if inactivated at the gene level (see also Chapter 12 for another approach to the problem of lethality). Since the power of the antisense approach first became obvious, studies focusing on the treatment of diseases have been initiated over the last few years. It seems only a matter of time before this strategy can be applied clinically to the treatment of particular diseases, e.g. infectious diseases like hepatitis B and C, or HIV infection, and even some forms of cancer. This chapter will give a brief overview of the different fields of medical application of antisense molecules, and will provide the reader with protocols for the experimental use of these molecules in cell culture and animal studies.

Deletions and point mutations in the gene encoding the cytoskeletal protein dystrophin and its isoforms cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy (BMD), largely depending on whether the reading frame is lost or maintained respectively. Frameshift mutations tend to result in a lack of dystrophin at the sarcolemma, destabilization of the membrane and degeneration of skeletal muscle. The mdx mouse is a valuable animal model of DMD as it bears a nonsense point mutation in exon 23 of the murine DMD gene leading to an absence of dystrophin expression in the muscle sarcolemma and muscular dystrophy. This report represents a novel approach to correct dystrophin deficiency at the post-transcriptional level by transfection of muscle cells with antisense RNA. Essentially, 2'- O -methyl oligoribonucleotides (2'OMeRNA) were delivered to the nuclei of primary mdx myoblasts in culture. Dystrophin expression was observed in the sarcolemma of transfected mdx myotubes after transfection by an oligonucleotide complementary to the 3' splice site of murine dystrophin intron 22. Direct sequencing of RT-PCR products from these cells revealed precise splicing of exon 22 to exon 30, skipping the mutant exon and creating a novel in-frame dystrophin transcript. As patients with comparable in-frame internal deletions show relatively mild myopathic symptoms, this may in the future offer a therapeutic approach for DMD, as well as for other inherited disorders.

Numerous chemically modified oligonucleotides have been developed so far and show their own unique chemical properties and pharmacodynamic/pharmacokinetic characteristics. Among all non-natural nucleotides, to the best of our knowledge, only five chemistries are currently being tested in clinical trials: phosphorothioate, 2´-O-methyl RNA, 2´-O-methoxyethyl RNA, 2´,4´-bridged nucleic acid/locked nucleic acid and the phosphorodiamidate morpholino oligomer. Since phosphorothioate modification can improve the pharmacokinetics of oligonucleotides, this modification is currently used in combination with all other modifications except phosphorodiamidate morpholino oligomer. For the treatment of metabolic, cardiovascular, cancer and other systemic diseases, the phosphorothioate class of drugs is obviously helpful, while superior efficacies can be observed in phosphorodiamidate morpholino oligomer compared to other classes of oligonucleotides for the treatment of Duchenne muscular dystrophy. Which properties of antisense molecules are actually essential for clinical applications? In this article, we provide an overview of the medicinal chemistry of existing non-natural antisense molecules, as well as their clinical applications, to discuss which properties of antisense oligonuculeotides affect therapeutic potency.

Antisense- and antigene-based drug design strategies in oncology

A reliable and specific modulation of gene expression in a cell would greatly facilitate the study of unknown gene functions, to combat pathogenic gene products or to control processes in biotechnology. One exciting option to achieve this goal is the application of antisense molecules such as antisense RNAs, antisense oligonucleotides or catalytic RNAs named ribozymes. All of these molecules bind to their target RNAs via complementary sequences, thereby forming base-paired duplices. Thus, in theory they permit a sequence-specific inhibition of gene expression. It is therefore not surprising that antisense approaches are increasingly used to understand the function of a given gene product, or to interfere with virus replication or cancerogenesis. Significant progress has been made in the design of antisense molecules, their delivery to cells, and in the understanding of their mode of action. However, compared to in vitro conditions, inside the cell the situation is much more complicated because of additional factors such as RNA-binding proteins and several intracellular compartments. Thus, in spite of the apparent simplicity of antisense approaches, many problems such as intracellular stability of the antisense molecule, target site selection, or colocalization of antisense molecule and target RNA remain to be solved. This review will address several of these problems and will discuss possible solutions. Due to space limitations, it is restricted to aspect of ribozyme applications.