Evaluation of off‐target effects of gapmer antisense oligonucleotides using human cells

Antisense oligonucleotide (ASO) has the potential to induce off‐target effects due to complementary binding between the ASO and unintended RNA with a sequence similar to the target RNA. Conventional animal studies cannot be used to assess toxicity induced by off‐target effects because of differences in the genome sequence between humans and other animals. Consequently, the assessment of off‐target effects with in silico analysis using a human RNA database and/or in vitro expression analysis using human cells has been proposed. Our previous study showed that the number of complementary regions of ASOs with mismatches in the human RNA sequences increases dramatically as the number of tolerated mismatches increases. However, to what extent the expression of genes with mismatches is affected by off‐target effects at the cellular level is not clear. In this study, we evaluated off‐target effects of gapmer ASOs, which cleave the target RNA in an RNase H‐dependent manner, by introducing the ASO into human cells and performing microarray analysis. Our data indicate that gapmer ASOs induce off‐target effects depending on the degree of complementarity between the ASO and off‐target candidate genes. Based on our results, we also propose a scheme for the assessment of off‐target effects of gapmer ASOs.     

Nucleotide sequence homology of thirteen tobamovirus RNAs as determined by hybridization analysis with complementary DNA

The extent of RNA sequence homology between 13 isolates of tobamoviruses has been estimated by hybridization analysis using radioactive complementary DNA. Hybrid formation between complementary [3H]DNA prepared against 7 of the tobamovirus RNAs and the RNAs of the 13 isolates was assayed with the single-strand-specific S1 nuclease. The presence of mismatched regions in the DNA: RNA hybrids was shown by variation of the stringency of the conditions for the hybridization and S1 nuclease assay. The data obtained have allowed the allocation of the 13 isolates into five groups on the basis of significant sequence homology between members of each group. Members of two of these groups were further divided into subgroups on the basis of differences in the hybridization data. Although hybridization analysis using labeled complementary DNA is considered the method of choice at present for estimating sequence homology between viral RNAs, the limitations inherent in any hybridization approach are discussed.     

Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene that result in a deficiency of SMN protein. One approach to treat SMA is to use antisense oligonucleotides (ASOs) to redirect the splicing of a paralogous gene, SMN2, to boost production of functional SMN. Injection of a 2'-O-2-methoxyethyl-modified ASO (ASO-10-27) into the cerebral lateral ventricles of mice with a severe form of SMA resulted in splice-mediated increases in SMN protein and in the number of motor neurons in the spinal cord, which led to improvements in muscle physiology, motor function and survival. Intrathecal infusion of ASO-10-27 into cynomolgus monkeys delivered putative therapeutic levels of the oligonucleotide to all regions of the spinal cord. These data demonstrate that central nervous system-directed ASO therapy is efficacious and that intrathecal infusion may represent a practical route for delivering this therapeutic in the clinic.     

HDR‐del: A tool based on Hamming distance for prioritizing pathogenic chromosomal deletions in exome sequencing

High‐density oligonucleotide arrays have widely been used to detect pathogenic chromosomal deletions. In addition to high‐density oligonucleotide arrays, programs using whole‐exome sequencing have become available for estimating copy‐number variations using depth of coverage. Here, we propose a new statistical method, HDR‐del, to prioritize pathogenic chromosomal deletions based on Hamming distance in exome sequencing. In vcf (variant call format) files generated from exome sequencing, hemizygous chromosomal deletion regions lack heterozygous variants and lead to apparent long runs of homozygosity (ROH). In our Hamming distance ratio (HDR)‐del approach, we calculate the “difference” in heterozygous status between an affected individual and control individuals using the HDR over all candidate chromosomal deletion regions defined as ROH longer than 1Mbp. Using a suitable test statistic, which is expected to be large for a true pathogenic deletion region, we prioritize candidate chromosomal deletion regions based on this statistic. In our approach, we were able to considerably narrow down true pathogenic chromosomal deletion regions, which were confirmed by high‐density oligonucleotide arrays in four mitochondrial disease patients. Our HDR‐del approach represents an easy method for detecting chromosomal deletions.     

An MLPA‐Based Strategy for Discrete CNV Genotyping: CNV‐miRNAs as an Example

Copy number variation (CNV) has become well recognized in recent years. It has been estimated that common CNVs account for approximately 10% of the human genome and that they overlap hundreds of genes and other functional genetic elements. Although substantial progress in genome‐wide CNV analysis has been made recently, there is still a need for a method that allows precise genotyping of selected CNVs. Here, we describe a novel strategy for CNV genotyping, taking advantage of the general principles of the multiplex ligation‐dependent probe amplification (MLPA) method and short oligonucleotide probes, allowing easy custom design and generation of assays for almost any genomic region of interest. As a proof‐of‐concept, we developed two assays covering 17 candidate CNV regions that overlap human miRNA genes. Extensive quality control analysis demonstrated high reproducibility and reliability of the genotypes determined using our method. Detailed analysis of identified CNVs revealed that they are highly differentiated among the HapMap populations. The main advantages of the developed strategy include the simplicity of the assay design, its flexibility in terms of the selection of genomic regions, and its low cost (<$1–$10/genotype, depending on scale of experiment). These advantages make the presented strategy attractive for large‐scale genetic analyses.     

Effect of trastuzumab-modified antisense oligonucleotide-loaded human serum albumin nanoparticles prepared by heat denaturation

Nanoparticles represent a promising tool for targeted drug delivery to tumour cells and are able to protect drugs against degradation. In our present study we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against Plk1 (polo-like kinase 1) prepared by heat denaturation instead of using glutaraldehyde. Glutaraldehyde can lead to an inactivation of ASOs through chemical crosslinking and is a toxic entity. We examined the ideal preparation conditions and characterised the resulting particles in terms of physico-chemical properties, ASO recovery after enzymatic degradation and stability. Stable monodisperse nanoparticles with an ASO recovery of more than 80% could be prepared at a temperature of 105 degrees C for 10 min. Furthermore we performed quantitative real-time PCR and Western blot to detect an ASO-mediated effect on Plk1 in BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression. Thus, this is the first report of ASO-loaded HSA nanoparticles prepared by heat denaturation, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells.     

OLIGSCAN: a computer program to assist in the design of PCR primers homologous to multiple DNA sequences.

OLIGSCAN (oligonucleotide scanner) is a computer program for IBM-PC-compatible computers that allows the user to scan up to 200 DNA sequences for homology to oligonucleotide sequences of interest. Once a core sequence of longer than the user-defined minimum length is found, the remainder of the oligonucleotide is compared to the corresponding positions of the larger sequence to identify matches or mismatches flanking the core region. This algorithm results in identification of the longest possible homologous regions first. The program was originally designed to assist in the identification of potential annealing sites for polymerase chain reaction (PCR) primers in the genomic DNA of related strains of viruses. However, it may also be used for more general pattern-identification purposes, including scanning for various sequence motifs of functional importance. We present the analysis of homology to an oligonucleotide primer in 16 complete genomic sequences of the human and simian immunodeficiency viruses.     

Discrimination between HLA-DR1 (DRB1*0101) and DR'Br' (DRB1*0103) using sequence-specific oligonucleotide probes

Serological identification of the HLA-DQw1(w5)-associated or HLA-DQw3(w7)-associated DR'Br' (DRB1*0103) allele cannot be accomplished in the presence of a second DQw1(w5)-positive or DQw3(w7)-positive haplotype, respectively. DNA-restriction fragment length polymorphism (RFLP) analysis assists in identification of DR'Br', though not in the presence of DR1. We describe an alternative or complementary method for identification of DR'Br' using two oligonucleotide probes which target HLA-DRB1 gene HV3 regions.     

RISC is a 5' phosphomonoester-producing RNA endonuclease

Gene silencing in the process of RNA interference is mediated by a ribonucleoprotein complex referred to as RNA-induced silencing complex (RISC). Here we describe the molecular mechanism of target RNA cleavage using affinity-purified minimal RISC from human cells. Cleavage proceeds via hydrolysis and the release of a 3'-hydroxyl and a 5'-phosphate terminus. Substitution of the 2'-hydroxyl group at the cleavage site by 2'-deoxy had no significant effect, suggesting that product release and/or a conformational transition rather than a chemical step is rate-limiting. Substitution by 2'-O-methyl at the cleavage site substantially reduced cleavage, which is presumably due to steric interference. Mutational analysis of the target RNA revealed that mismatches across from the 5' or the 3' end of the siRNA had little effect and that substrate RNAs as short as 15 nucleotides were cleaved by RISC.     

Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR

BackgroundDirect immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses.ObjectivesEstablish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity.Study designNucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples.ResultsN gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses.ConclusionsAn emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy.     

Silencing disease genes in the laboratory and the clinic

Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past 20 years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology.     

Rapid differentiation of avian infectious bronchitis virus isolates by sample to residual ratio quantitation using real-time reverse transcriptase-polymerase chain reaction

A rapid diagnostic assay for differentiating avian infectious bronchitis virus (IBV) isolates was developed. The basis of the assay is the cleavage of target RNA by RNase H mediated by sequence-specific chimeric oligonucleotides followed by sample to residual ratio quantitation (SRRQ) using RRT-PCR. Four serotype-specific chimeric oligonucleotides were designed, one each for the Massachusetts, Connecticut, Arkansas, and Delaware/Georgia 98 serotypes, and tested for their ability to mediate specific cleavage of target RNA from known homologous and heterologous strains of IBV. Specific cleavage of target RNAs by each chimeric oligonucleotide was verified using agarose gel analysis and RRT-PCR. There were no non-specific cleavage products. Eight different IBV strains representing seven serotypes were tested and each chimeric oligonucleotide mediated cleavage of target RNA only from strains within the serotype that the chimeric was designed against. The SRRQ assay was evaluated on 15 samples without prior knowledge of their grouping and correctly identified the serotype of each sample. The assay is rapid; six samples can be tested in approximately 4 h. In addition, the primer set amplifies all IBV RNAs tested to date and provides a built in control for detecting IBV whether it is typeable or not.     

MicroRNA在人类肿瘤中的角色及其应用

微小RNA（microRNAs,miRNAs）是一类含有约22个核苷酸的内源性非编码RNAs,通过与靶基因mRNA的3′端非翻译区（3′untranslated region,3′UTR）不完全性互补配对,介导转录后基因调控。miRNAs在人类肿瘤的发生和发展中扮演重要角色,发挥着癌基因或抑癌基因的作用。miRNAs可以作为肿瘤的生物标记和诊断工具,将成为新一代的抗肿瘤治疗的靶点。