Sequence-specific Interference Research Articles

Introduction to Gene Therapy

After many years of research and numerous setbacks, there are now undeniable success stories of gene therapies, namely the direct modification of genetic information on the DNA or RNA level. Both "ex vivo" strategies, i. e. the genetic manipulation of patient cells in a dish, as well as "in vivo" approaches are being used successfully. In addition to the supplementation of defective genes, the use of the CRISPR-Cas9 system to alter nuclear DNA sequences and the sequence-specific interference with the transcriptional process on the RNA level can be designated as gene therapies in a broad sense.

Sequence-specific error profile of Illumina sequencers

We identified the sequence-specific starting positions of consecutive miscalls in the mapping of reads obtained from the Illumina Genome Analyser (GA). Detailed analysis of the miscall pattern indicated that the underlying mechanism involves sequence-specific interference of the base elongation process during sequencing. The two major sequence patterns that trigger this sequence-specific error (SSE) are: (i) inverted repeats and (ii) GGC sequences. We speculate that these sequences favor dephasing by inhibiting single-base elongation, by: (i) folding single-stranded DNA and (ii) altering enzyme preference. This phenomenon is a major cause of sequence coverage variability and of the unfavorable bias observed for population-targeted methods such as RNA-seq and ChIP-seq. Moreover, SSE is a potential cause of false single-nucleotide polymorphism (SNP) calls and also significantly hinders de novo assembly. This article highlights the importance of recognizing SSE and its underlying mechanisms in the hope of enhancing the potential usefulness of the Illumina sequencers.

New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus

Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene of the viral hemorrhagic septicemia virus (VHSV), a rhabdovirus affecting fish. Eight siRNA sequences were first designed, synthesized and screened for inhibition of in vitro VHSV infectivity. Small hairpin (sh) DNAs corresponding to three selected siRNAs were then cloned into pRNA-CMV3.1/puro plasmids, transfected into EPC cells and transformed clones were obtained by puromycin selection. Sequence-specific interference with VHSV could only be observed with EPC clones transformed with a mixture of the three shDNAs, rather than with those clones obtained with individual sh DNAs. However, interference was not specific for VHSV as infection with an heterologous fish rhabdovirus, was also reduced to a similar extent. It was shown that this reduction was not due to an Mx response in the transformed cell clones. Here, we discuss some of the possible reasons for such data and future work directions. EPC clones stably expressing rhabdoviral specific siRNA sequences could be a strategy to further investigate the use of RNA interference for targeting costly fish pathogenic viruses.

Engineered virus-encoded pre-microRNA (pre-miRNA) induces sequence-specific antiviral response in addition to nonspecific immunity in a fish cell line: Convergence of RNAi-related pathways and IFN-related pathways in antiviral response

Transfection with synthesized virus-specific small interfering RNAs (siRNAs) efficiently inhibits viral replication in viral-infected fish cell lines, implying the involvement of RNA interference (RNAi)-related pathways in the antiviral response of fish cells. Here, we demonstrate that plasmid expressing virus-encoded pre-microRNAs (pre-miRNAs) can also inhibit viral replication through these pathways. By incorporating sequences encoding miRNAs specific to major capsid protein (MCP) gene of red sea bream iridovirus (RSIV) and a miRNA specific to hirame rhabdovirus (HIRRV) genome into a murine miR-155 pre-miRNA backbone, we were able to intracellularly express viral pre-miRNAs (miR-MCPs and miR-HIRRV) in a fish cell line. The miR-MCPs and miR-HIRRV, delivered as pre-miRNA precursors in transfected cells, inhibited viral replication when these cells were infected with the target virus. Although this may suggest sequence-specific interference, inhibitory effect on viral replication was also observed in cells transfected with a plasmid expressing pre-miRNA targeting β-galactosidase gene (miR-LacZ) that served as a specificity control. Expression of pre-miRNAs was found to activate interferon (IFN)-related pathways, correlating with upregulation of the antiviral IFN-induced Mx protein. The antiviral effects of viral-miRNAs observed here were partly the result of the antiviral miRNA-related pathways and partly the result of the antiviral IFN-related pathways. We propose that engineered virus-encoded pre-miRNA can engage not only RNAi-related pathways but also IFN-related pathways to induce potent antiviral responses in fish cells.

Titers of HIV-based Vectors Encoding shRNAs are Reduced by a Dicer-dependent Mechanism

Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful in deciphering gene function, and are being considered for therapeutic knockdown of target genes in humans. We constructed HIV-based vectors encoding shRNA against HIV coreceptor chemokine (C-C motif) receptor 5 (CCR5). Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers than those of the empty vector. Co-transfection of expression plasmids encoding CCR5 in the producer cells yielded a tenfold increase in viral titer, thereby indicating that CCR5 mRNA, rather than HIV vector mRNA, could be the target of CCR5 shRNA. Similar increases in vector titer were observed after the H1 promoter was deleted. When Nodamura-virus B2 protein or Adenovirus VA.1 RNA (inhibitors of the Dicer-dependent siRNA pathway) were added to the producer cells, the vector titer rose almost to the level of that of the empty vector. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) suggested that the effects were in part caused by reduction in vector RNA in the producer cells. Similar results were observed with a retroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism which, to a large extent, can be reversed by inhibiting that pathway. This may have important implications for large-scale production of RNA vectors encoding shRNAs.

Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide.

Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5–10 μM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 μM concentration of the R6Pen–PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen–PNA705 structure–function relationship have also been evaluated.

Sequence-specific interference by small RNAs derived from adenovirus VAI RNA

A virus-associated RNA (VAI) of adenoviruses is a cytoplasmic non-coding RNA and it plays an important role for viral replication in infected cells. VAI RNA transcripts, produced by RNA polymerase III (pol III), form tightly structured stems, which confer resistance to cellular defense systems. We demonstrate here that small RNAs of approximately 22 nucleotides are produced from a terminal stem region but not from an apical stem of VAI RNA. We determined the processing sites of VAI RNA by S1 nuclease mapping and further confirmed that the processed small RNA can act as small interfering RNAs (siRNAs) or as microRNAs (miRNAs) in transient transfection assays and during viral infection. Our data demonstrate that non-coding RNAs synthesized by pol III can be substrates for Dicer, and diced small RNAs might regulate cellular phenomena as siRNAs and miRNAs.

SiRNA injection induces sequence-independent protection in Penaeus monodon against white spot syndrome virus

White spot syndrome virus (WSSV) is a major disease in crustaceans, particularly shrimp, due to the current intensity of aquaculture practices. Novel strategies including vaccination to control this virus would be highly desirable. However, invertebrates lack a true adaptive immune response system and seem to rely on various innate immune responses. An alternative and more specific approach to counteract WSSV infections in shrimp could be by the exploitation of RNA interference. As long dsRNA molecules induce a general, sequence-independent anti-viral immunity in shrimp [Robalino, J., Browdy, C.L., Prior, S., Metz, A., Parnell, P., Gross, P., Warr, G., 2004. J. Virol. 78, 10442–10448], it was investigated whether shorter 21 nt siRNAs with homology to the WSSV vp15 and vp28 genes would give a sequence-specific interference response in the shrimp Penaeus monodon. Vp28 siRNAs as well as nonspecific control gfp siRNAs were able to specifically and efficiently silence their homologous genes in a heterologous baculovirus insect cell expression system. However, in shrimps no such a specific effect was observed. Shrimp injected with vp15 or vp28 siRNAs before WSSV challenge gave a significantly lower mortality rate, but not significantly different when shrimps were injected with gfp siRNA. Thus, large dsRNA molecules as well as siRNAs induce a sequence-independent anti-viral immunity when injected in shrimp.

Specific interference with gene expression and gene function mediated by long dsRNA in neural cells

Double-stranded (ds) RNA-induced sequence-specific interference with gene expression, RNA interference (RNAi), has been extensively used in invertebrates, allowing for efficient and high-throughput gene silencing and gene function analysis. In vertebrates, however, use of RNAi to study gene function has been limited due to non-specific effects induced by double-stranded RNA (dsRNA)-dependent protein kinase and interferon activation. dsRNA-induced specific inhibition of vertebrate gene expression has only been shown in embryonic and non-differentiated mammalian cells. In this report, we demonstrate dsRNA-induced specific interference of gene expression and gene function in partially as well as fully differentiated mouse neuroblastoma cells. Specific silencing was observed in the expression of an integrated transgene coding for green fluorescent protein and a variety of endogenous genes. Moreover, we show that RNAi-mediated inhibition of poly (ADP-ribose) polymerase (PARP) expression induced cellular resistance to oxygen–glucose deprivation, consistent with the role of PARP in ischemia-induced brain damage. Our results indicate that RNAi can be used as a powerful tool to study gene function in neural cells.

Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells.

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of Escherichia coli RNase III to cleave double-stranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in Drosophila S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells. esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.

Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation.

Conditions for the electroporation of mouse oocytes and preimplantation embryos have been optimised by following the incorporation of rhodamine labeled dextran. This procedure includes a step to weaken but not remove the zona pellucida that helps achieve good survival. This approach has been applied to introduce double-stranded RNA for c-mos into oocytes and green fluorescent protein (GFP) into transgenic GFP-expressing embryos at the 1- and 4-cell stages. In both cases we were able to observe sequence-specific interference with the expression of the target gene--a failure of oocytes to arrest at metaphase II and a loss in the green fluorescence of embryos by the morula or blastocyst stages. These effects could be observed in multiple oocytes or embryos allowed to develop together following electroporation.

Technical advance. Double-stranded RNA interferes with gene function at the single-cell level in cereals.

Double-stranded RNA (dsRNA) has been shown to specifically interfere with gene function in several organisms including tobacco and the model plant Arabidopsis. Here, we report on rapid and sequence-specific interference of dsRNA with gene function in cereals. Delivery of cognate dsRNA into single epidermal cells of maize, barley or wheat by particle bombardment interfered with the function of co-bombarded UidA (GUS) and TaGLP2a:GFP reporter genes. Cognate dsRNA was also found to specifically interfere with the function of the endogenous genes A1 and Ant18 encoding dihydroflavonol-4-reductase in maize and barley, respectively. Dihydroflavonol-4-reductase is an essential enzyme of the anthocyanin biosynthetic pathway in maize and barley. This pathway can be induced by transient expression of the C1- and b-Peru genes that encode transcription factors. In the presence of dsRNA corresponding to the dihydroflavonol-4-reductase gene, C1- and b-Peru-dependent, cell-autonomous accumulation of red anthocyanin pigments in bombarded cells of maize and barley was reduced. dsRNA was also demonstrated to negatively interfere with Mlo, which encodes a negative regulator of race non-specific resistance to the powdery mildew fungus in barley. In the presence of Mlo dsRNA, transformed cells became more resistant, thereby phenocopying plants that carry a heritable loss-of function mlo resistance allele. The results suggest that direct delivery of dsRNA to cereals leads to a rapid and sequence-specific interference with gene function at the single-cell level.