Epistemic_Statements  

CORD-19:d1ebf10b568022cf6500990bcfadf9054864211b JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
T1 272-451 Epistemic_statement denotes Numerous pre-clinical studies are evaluating RNAi as a novel therapeutic modality in the battle against gain-of-function autosomal dominant diseases, cancer, and viral infections.
T2 452-604 Epistemic_statement denotes One emerging concern is that RNAi mono-therapies might ultimately fail to control viruses that can escape silencing by mutation and/or RNAi suppression.
T3 605-778 Epistemic_statement denotes Thus, sophisticated strategies are being developed that aim to avert viral resistance by combining RNAi effectors with each other or with further gene expression inhibitors.
T4 779-1002 Epistemic_statement denotes Several reports already validate this new concept of "combinatorial RNAi" (coRNAi) and illustrate its versatility by describing co-expression of RNAi triggers directed against single or multiple, viral or cellular, targets.
T5 1003-1115 Epistemic_statement denotes Other studies document the successful delivery of these triggers with additional RNA-or protein-based silencers.
T6 2190-2378 Epistemic_statement denotes Moreover, approximately 39 million people worldwide were living with human immunodeficiency virus (HIV) in 2005, with approximately 4 million new infections and 3 million deaths that year.
T7 2535-2725 Epistemic_statement denotes Global pandemics caused by newly emerging viral infections, such as Ebola, severe acute respiratory syndrome coronavirus, and avian influenza (H5N1), present further threats to human health.
T8 2726-2832 Epistemic_statement denotes The reasons for the persistence of human viruses and the emergence of new infectious diseases are complex.
T9 2833-3022 Epistemic_statement denotes Key is the extensive variation and flexibility of viral genomes, resulting from a combination of minimal generation times, notoriously inaccurate reproduction, and intra-host recombination.
T10 3140-3374 Epistemic_statement denotes This particularly applies to RNA viruses such as HCV, whose RNA-dependent RNA polymerase incorporates the extreme number of 10 3 mutations per viral nucleotide per year (or eight per genome, 100-fold higher than for HBV, a DNA virus).
T11 3739-3984 Epistemic_statement denotes Further shifting the balance of power is the fact that many viruses exist in genetically distinct quasi-species and subtypes and/or have developed "stealth and cunning" mechanisms to out-maneuver or evade the innate and adaptive immune response.
T12 3985-4095 Epistemic_statement denotes 4 Unfortunately, our existing treatment options for viral infections are usually ineffective and very limited.
T13 5050-5144 Epistemic_statement denotes Fire and Craig C. Mello), RNAi is a natural phenomemon of gene silencing by small duplex RNAs.
T14 5648-5820 Epistemic_statement denotes 9 Following expression as long pri-miRNAs, they are processed by the nuclear enzyme Drosha into shorter pre-miRNAs and then transported into the cytoplasm (via Exportin-5).
T15 5821-5996 Epistemic_statement denotes There, the adenosine triphosphate-dependent RNAse III-like Dicer enzyme generates even shorter (approximately 21 nt) double-stranded RNAs, the small interfering RNAs (siRNAs).
T16 6205-6316 Epistemic_statement denotes 10 The extreme efficiency and specificity of this process make RNAi highly attractive for anti-viral therapies.
T17 6766-6946 Epistemic_statement denotes However, their use might naturally be limited to mucosal tissues or localized and accessible sites of viral infection (e.g., the respiratory and female genital tracts and the eye).
T18 7173-7256 Epistemic_statement denotes 14 The latter application is currently being evaluated in a phase I clinical trial.
T19 7257-7423 Epistemic_statement denotes Conversely, a gene therapy approach involving delivery of RNAi expression cassettes is more appropriate for treatment of chronic infections such as HBV, HCV, and HIV.
T20 7424-7608 Epistemic_statement denotes 1 Typically, the trigger is a miRNAlike sequence (derived from a natural miRNA 15 or an artificial short hairpin RNA (shRNA)) under the control of an RNA polymerase II or III promoter.
T21 7609-7772 Epistemic_statement denotes These cassettes are small and thus readily incorporated into any of the established gene therapy vectors, such as lentiviruses and AAVs (adeno-associated viruses).
T22 7957-8103 Epistemic_statement denotes 16 Another general advantage of RNAi as an anti-viral therapy is that triggers with perfect viral sequence complementarity induce target cleavage.
T23 8326-8476 Epistemic_statement denotes 11, 12 Last but not least, RNAi silencing requires a minimal target of only 19-21 nt, which might be sufficient to co-suppress related viral isolates.
T24 9131-9189 Epistemic_statement denotes This has already been illustrated by a variety of reports.
T25 9221-9352 Epistemic_statement denotes noted that prolonged incubation of poliovirusinfected, siRNA-treated cells resulted in enrichment of an RNAiresistant point mutant.
T26 9963-10020 Epistemic_statement denotes 23 HIV's propensity to escape was confirmed by Das et al.
T27 10118-10150 Epistemic_statement denotes Interestingly, Westerhout et al.
T28 10880-11081 Epistemic_statement denotes This should not be surprising, as double-stranded RNA is often generated during viral replication, exposing the virus to host RNAi and likely exerting pressure to evolve an anti-RNAi counter-mechanism.
T29 11465-11606 Epistemic_statement denotes 29 However, their main function is inhibition of protein kinase R, and RNAi suppression has been shown only in non-vertebrate cells thus far.
T30 12274-12450 Epistemic_statement denotes 35 On the other hand, it is striking to note that some human viruses hijack the RNAi machinery to carry out their own replication strategies, which might seem counterintuitive.
T31 12451-12575 Epistemic_statement denotes [26] [27] [28] One remarkable example is again HCV, which subverts a liver-specific miRNA (miR-122) for its gene expression.
T32 12576-12762 Epistemic_statement denotes 36 Moreover, other viruses were recently found to encode their own miRNAs, albeit in most cases (e.g., HIV or Epstein-Barr virus) without evidence for functional or genetic significance.
T33 13002-13004 Epistemic_statement denotes 42
T34 13005-13141 Epistemic_statement denotes As our knowledge of the interactions between viruses and the RNAi pathway expands, it will continue to shape our therapeutic strategies.
T35 13634-13805 Epistemic_statement denotes The emerging solution to thwart viral evolution and circumvent the related issues is to multiplex RNAi triggers or to combine them with other silencers of gene expression.
T36 14989-15130 Epistemic_statement denotes Such a mix of RNAi and unrelated silencers will also minimize the potential risks associated with high-level mi/shRNA expression in the cell.
T37 15407-15499 Epistemic_statement denotes We also include a few examples for functional genomics and treatment of non-viral disorders.
T38 15599-15736 Epistemic_statement denotes Examples include a study by Kronke et al., who used a library of endoribonuclease-prepared siRNAs to block HCV replicons in cell culture.
T39 16167-16182 Epistemic_statement denotes [45] [46] [47]
T40 16183-16322 Epistemic_statement denotes Concatemerization of single RNAi triggers (Figure 1a) represents the simplest example of coRNAi and has been reported sporadically to date.
T41 16323-16502 Epistemic_statement denotes Intended to raise intra-cellular sh/miRNA levels, it might be especially useful against viral targets that replicate and spread at high enough rates to out-compete low-level RNAi.
T42 17082-17097 Epistemic_statement denotes Gonzalez et al.
T43 18581-18668 Epistemic_statement denotes Instead, the authors noted decreased RNAi from the tandem plasmid, for reasons unclear.
T44 18859-19022 Epistemic_statement denotes 50 Plasmids expressing anti-SOD2 mIR-30 from a ubiquitin C promoter were injected into fertilized eggs, yielding two lines carrying a single or three miRNA copies.
T45 19023-19106 Epistemic_statement denotes The singlecopy line had higher siRNA levels, correlating with better SOD knockdown.
T46 19107-19239 Epistemic_statement denotes Crossing both lines yielded bigenic heterozygous mice expressing even higher siRNA levels and showing a near SOD knockout phenotype.
T47 19438-19448 Epistemic_statement denotes Sun et al.
T48 19449-19583 Epistemic_statement denotes concatenated mIR-30-based anti-S-phase kinaseassociated protein 2 or anti-androgen receptor hairpins under a cytomegalovirus promoter.
T49 19771-19874 Epistemic_statement denotes However, addition of a third copy yielded only a marginal further (disproportionally smaller) increase.
T50 20279-20417 Epistemic_statement denotes 52 Concatemerization of up to eight anti-luciferase miRNAs under a single ubiquitin C promoter resulted in a progressive increase in RNAi.
T51 21169-21361 Epistemic_statement denotes Together, these few studies already clearly highlight the feasibility of expressing multiple sh/miRNAs from a single construct, although there exist few therapeutic applications at this point.
T52 21362-21552 Epistemic_statement denotes Vectors employing miRNAs appear to be preferred for expression of multiple hairpins as they allow the use of a single promoter, which offers the potential for spatio-temporal coRNAi control.
T53 21955-22367 Epistemic_statement denotes In brief, there were two different goals: (i) to establish coRNAi as a surrogate genetic tool for basic studies, allowing the dissection of overlapping functions of individual factors to biochemical pathways; (ii) more relevant in the context of this review, to elucidate the usefulness of coRNAi for the treatment or prevention of viral infection and escape by co-targeting multiple viral and/or cellular genes.
T54 22378-22514 Epistemic_statement denotes were among the first to apply a coRNAi approach to the study of gene function, via simultaneous inhibition of multiple endogenous mRNAs.
T55 22515-22713 Epistemic_statement denotes 53 They designed two shRNAs (under separate U6 promoters) to target the αand β-isoforms of glycogen synthase kinase 3, two related enzymes involved in various cellular processes and human disorders.
T56 22714-22918 Epistemic_statement denotes In stably co-transfected cells, coRNAi of both isoforms led to an additive increase in expression of the glycogen synthase kinase 3 target β-catenin, as compared with inhibition of the individual enzymes.
T57 23716-23942 Epistemic_statement denotes Similar to the results of Gonzalez et al., 48 shRNA expression and Smad knockdown could be maintained for at least 20 passages, likely owing to the small shRNA number and the low expression levels from the integrated plasmids.
T58 23943-24168 Epistemic_statement denotes As hoped, phenotypic analyses of their various cell lines revealed different contributions of all three Smads to parameters such as wound closure and cell migration, providing further insight into the role of Smads in cancer.
T59 24600-24747 Epistemic_statement denotes 56 In detail, the group engineered lentiviruses conditionally to express two mIR-30 hairpins targeting the heterotrimeric G proteins Gα12 and Gα13.
T60 24748-24952 Epistemic_statement denotes Analyses of reporter gene expression (luciferase fused with a serum response element) allowed them to delineate a specific role of Gα13 of transmitting receptor-mediated serum response element activation.
T61 24953-25098 Epistemic_statement denotes This study is thus another illustration of coRNAi as a powerful experimental platform for analysis of potential redundancy in signaling pathways.
T62 25099-25202 Epistemic_statement denotes From a clinical standpoint, two of the most interesting targets for therapeutic coRNAi are HCV and HIV.
T63 25909-26120 Epistemic_statement denotes used adenovirally delivered, U6-driven shRNAs against cellular La, polypyrimidine tract-binding protein, and human vesicle-associated membrane protein-associated protein of 33 kd, all known to interact with HCV.
T64 26512-26670 Epistemic_statement denotes However, a fundamental concern with this strategy is that knocking down endogenous genes could create an unacceptable loss-of-function pathology for the cell.
T65 26810-27147 Epistemic_statement denotes This is exemplified by a report by Korf et al., who co-targeted the two cellular HCV co-factors HuR (Hu antigen R, binds to the HCV 3 -untranslated region, resulting in its stabilization) and PSMA7 (proteasome α-subunit 7, modulates HCV-internal ribosome entry site activity), together with the HCV genome (5 -or 3 -untranslated region).
T66 28222-28414 Epistemic_statement denotes 59 In HCV replicon cells, all individual shRNAs (under separate H1 promoters, delivered by a lentivirus) efficiently reduced replication or expression of their specific target by at least 80%.
T67 28415-28555 Epistemic_statement denotes Similar results (with respect to the individual targets) were obtained for coRNAi vectors expressing two or all three shRNAs simultaneously.
T68 28556-28694 Epistemic_statement denotes Important conclusions were the lack of competition among the individual shRNAs and the absence of non-specific effects from their vectors.
T69 29578-29768 Epistemic_statement denotes 60 Accordingly, the authors used a plasmid encoding a 51-bp-long, U6-driven shRNA for the efficient co-targeting of the NS5B gene from two distinct HCV strains differing in nine nucleotides.
T70 29769-29903 Epistemic_statement denotes Compared with a conventional 20-mer shRNA, the longer hairpin not only suppressed both isolates but also yielded more rapid knockdown.
T71 29904-30105 Epistemic_statement denotes Although this was not strictly a coRNAi approach, this study is notable because the results implied the generation of multiple different siRNAs from the long precursor (albeit not truly characterized).
T72 30269-30281 Epistemic_statement denotes Boden et al.
T73 32097-32232 Epistemic_statement denotes Virus challenge assays showed a marked protection of the transfected cells from HIV, in particular by the combined CXCR4/CD4 construct.
T74 32945-33119 Epistemic_statement denotes One concern is that inhibiting a specific receptor may select for viral variants that use a non-targeted, different (co-)receptor, ultimately negating any therapeutic effect.
T75 33235-33354 Epistemic_statement denotes 62 In contrast, CCR5 might be dispensable for life, as there are asymptomatic individuals homozygous for CCR5 mutation.
T76 33465-33618 Epistemic_statement denotes Nevertheless, CXCR4 knockdown is critical in stem cells (a major target for HIV therapies) as this molecule plays a role in cell homing into bone marrow.
T77 33717-33729 Epistemic_statement denotes Chang et al.
T78 33821-34066 Epistemic_statement denotes 65 A critical finding was that a combination of three vectors, directed against highly conserved regions in the viral pol, int, and vpu genes, outperformed the individual shRNAs in terms of suppressing HIV in a stable virus-producer T-cell line.
T79 34067-34197 Epistemic_statement denotes Despite the increased efficacy, the possible formation or prevention of escape mutants was not evaluated in this short-term study.
T80 34198-34294 Epistemic_statement denotes It was instead addressed in a more comprehensive, very recent study by ter Brake and colleagues.
T81 34295-34436 Epistemic_statement denotes 66 In a screen of all HIV-1 subtypes (including the LAI prototype) for highly conserved regions, the authors identified 19 potential targets.
T82 34437-34549 Epistemic_statement denotes Of a battery of 86 shR-NAs against these targets, 21 were found to be transiently effective from an H1 promoter.
T83 34769-34878 Epistemic_statement denotes Intriguingly, all three shRNAs combined in one lentiviral vector conferred nearresistance to viral infection.
T84 34879-35019 Epistemic_statement denotes Most important, the group also studied the emergence of viral resistance in cells expressing only one or two of their most effective shRNAs.
T85 35020-35179 Epistemic_statement denotes As hoped, viral inhibition was more durable in the coRNAi cell line, although eventually (day 22) most cultures were positive, regardless of shRNA copy number.
T86 35180-35391 Epistemic_statement denotes Although they do not show the data, the authors also mentioned an improved vector expressing four different shRNAs (from separate and distinct promoters) and able to further delay viral escape for up to 60 days.
T87 35533-35696 Epistemic_statement denotes It is also particularly noteworthy that the shRNAs were carefully chosen to concurrently target all HIV-1 subtypes, although this was not confirmed experimentally.
T88 35697-35915 Epistemic_statement denotes Important for future use of this particular system will be to investigate the genetic stability of the threefold or fourfold shRNA lentiviral vectors, as well as the potential side effects from these unique constructs.
T89 36105-36308 Epistemic_statement denotes 67 When transduced via lentiviral vectors into CD4 + T cells, the individual shRNAs showed a more potent inhibitory effect on HIV replication than an anti-tat construct (the same one used by Boden et al.
T90 36309-36315 Epistemic_statement denotes 23 ) .
T91 36316-36519 Epistemic_statement denotes Remarkably, at a higher HIV dose where the single shRNAs were no longer able to control the virus, a combination of the intand att-specific shRNA vectors still gave strong suppression for almost 3 weeks.
T92 36520-36658 Epistemic_statement denotes In line with previous work, the group noted the emergence of resistant point mutants after HIV infection of single shRNA-expressing cells.
T93 36659-36857 Epistemic_statement denotes Subsequently generated shRNAs specific for these mutants could suppress their replication, but, unexpectedly, a combination of wild-type and mutant shRNAs had less effect on preventing viral escape.
T94 36858-36997 Epistemic_statement denotes The authors hypothesized intra-cellular competition of the various shRNA vectors for the same target site, but this idea was not validated.
T95 36998-37036 Epistemic_statement denotes As in the HCV studies by Akashi et al.
T96 37037-37164 Epistemic_statement denotes 60 and Watanabe et al., 61 the group also tested a long (50-nt) hairpin RNA covering the target region of their anti-int shRNA.
T97 37165-37322 Epistemic_statement denotes Interestingly, this construct was able to co-suppress both wild-type and mutant HIV strains, but the effect was weak and only transient, for reasons unknown.
T98 37323-37581 Epistemic_statement denotes Nonetheless, these articles together suggest that when the technical problems have been overcome and the safety of long hairpins can be guaranteed, combining multiple short and long RNAs might further increase the power of coRNAi to control viral resistance.
T99 37582-37701 Epistemic_statement denotes Collectively, the studies reviewed above clearly validate the promise of coRNAi to suppress viral infection and escape.
T100 37702-37847 Epistemic_statement denotes However, they also provide evidence for potential setbacks from co-expression of multiple hairpin RNAs in the same cell and from the same vector.
T101 37848-37969 Epistemic_statement denotes The issues include genetic instabilities, promoter or hairpin interference, and toxic side effects (see also Conclusion).
T102 38517-38701 Epistemic_statement denotes As hoped for, the coRNAi approach produced synergistic effects in terms of P2X 3 knockdown in cultured cells, but, interestingly, only when both agents targeted non-homologous regions.
T103 38702-38842 Epistemic_statement denotes The reasons for this competition remain elusive, but the findings are reminiscent of the study by Nishitsuji and colleagues described above.
T104 38843-39032 Epistemic_statement denotes 67 It is unclear whether the molecular mechanisms are related or even identical, but these two studies certainly prompt caution in attempts to target a single site with multiple inhibitors.
T105 39033-39047 Epistemic_statement denotes Jarczak et al.
T106 39048-39154 Epistemic_statement denotes were among the first to suggest a combination of shRNAs with hammerhead ribozymes (Rzs) for HCV treatment.
T107 39155-39305 Epistemic_statement denotes 69 The group targeted the highly conserved 5 -and 3 -viral untranslated regions with various U6-driven shRNAs or Rzs (under U6 or tRNA Val promoters).
T108 39306-39460 Epistemic_statement denotes After individual transfection into HCV replicon cell lines, their best candidates inhibited HCV NS5B expression by approximately 30% (Rzs) or 50% (shRNA).
T109 39461-39741 Epistemic_statement denotes Although mixing various Rzs increased overall inhibition marginally, it was most notable that combining the best Rzs and shR-NAs gave an approximately 25% additive effect to each shRNA, irrespective of its initial potential (however, combinations of shRNAs alone were not tested).
T110 39742-39929 Epistemic_statement denotes This study is an important proofof-concept, in particular as it further confirms the need to target different sites in the viral genome for maximum efficacy, in line with Watanabe's work.
T111 40112-40263 Epistemic_statement denotes At this point, relevant anti-HIV coRNAi approaches have been mostly reported by John Rossi's group, which is currently also preparing a clinical trial.
T112 40437-40455 Epistemic_statement denotes Instead, Li et al.
T113 40456-40704 Epistemic_statement denotes 64 used an anti-CCR5 ribozyme (driven by an adenoviral VA1 promoter) together with a trans-activation response region decoy (U6-promoted and embedded in small nucleolar RNA U16, to ensure co-localization with HIV Tat in nucleoli of infected cells).
T114 40705-40874 Epistemic_statement denotes The resulting vector provided a substantially greater survival advantage to HIV-challenged primary T or CD34 + stem cells compared with the individual ribozyme or decoy.
T115 40875-41091 Epistemic_statement denotes An shRNA against rev and tat tested in parallel was shown to reduce HIV p24 substantially in the same cell types, over the same period, but the results were not directly compared and the constructs were not combined.
T116 41092-41269 Epistemic_statement denotes Construct combination was reported in a follow-up study in which Rossi's group presented a lentiviral vector combining all three inhibitors from their earlier work (Figure 1e) .
T117 41604-41769 Epistemic_statement denotes Importantly, there was no evidence for evolution of escape mutants with the triple vector, although additional validation might be needed in view of the assays used.
T118 42235-42378 Epistemic_statement denotes Nevertheless, the overall impressive results with this unique and ingenious construct clearly illustrate the power of coRNAi for HIV therapies.
T119 42379-42624 Epistemic_statement denotes Consequently, a slightly modified vector (deleted for a gfp marker gene) will soon be tested in a clinical trial using autologous hematopoietic stem cells from acquired immunodeficiency syndrome/lymphoma patients and bone marrow transplantation.
T120 43197-43315 Epistemic_statement denotes created a lentiviral vector in which an anti-rev shRNA was expressed from an HIV-inducible RNA polymerase II promoter.
T121 43499-43707 Epistemic_statement denotes In stably transduced and HIV-challenged T cells, the double vector mediated 90% inhibition of HIV p24 protein and a high cell survival rate (although a direct comparison with single vectors was not provided).
T122 44100-44278 Epistemic_statement denotes 70 Last but not least, in addition to co-expression of transdominant anti-viral proteins, one can also envision synergistic gene silencing/addition in other therapeutic contexts.
T123 44279-44321 Epistemic_statement denotes One remarkable example by Samakoglu et al.
T124 45388-45639 Epistemic_statement denotes It is emerging as a powerful modality to battle some of the most notoriously challenging clinical targets (HCV, HIV, and other human viruses), and initial studies also affirm its great potential for treatment of metabolic or blood disorders or cancer.
T125 45640-45780 Epistemic_statement denotes Concurrently, coRNAi is quickly exceeding our expectations for its use in the study of basic processes, such as signaling or transformation.
T126 46074-46279 Epistemic_statement denotes The safety concern is based on a plethora of earlier reports on unexpected and adverse side effects from mono-RNAi treatments, including "off-target" silencing, IFN responses, and translational inhibition.
T127 46280-46451 Epistemic_statement denotes 73 Although it is obvious that these risks may increase proportionally with a coRNAi approach, another specific concern is oversaturation of the endogenous RNAi machinery.
T128 46452-46612 Epistemic_statement denotes This might at least result in competitive reduction of the effects of the individual silencers, which could indeed explain some of the findings described above.
T129 48419-48641 Epistemic_statement denotes Unfortunately, no published work has yet defined the limit to the number of exogenous hairpin RNAs that can be effectively incorporated into a (co)RNAi treatment, and it will likely vary with the types of cells and organs.
T130 49119-49330 Epistemic_statement denotes 78 Some exciting recent examples include promoters that are specifically activated in HCV-or HIV-infected cells 79, 80 or can be epigenetically and reversibly controlled using exogenous drugs or small molecules.
T131 49331-49504 Epistemic_statement denotes 81, 82 The use of these alternative promoters might concurrently help to circumvent the second concern with coRNAi, i.e., genetic instability of the multi-component vectors.
T132 49505-49827 Epistemic_statement denotes Although it is technically feasible to accommodate multiple sh/miRNA cassettes into virtually any present viral vector (including the smallest of all, AAV), there are hardly any data at this point on the likely risks of recombination or deletion caused by sequence similarities or identities among the individual elements.
T133 49828-50037 Epistemic_statement denotes Anecdotal evidence suggests this problem exists and could hamper the approach, as it might, for instance, explain the reported difficulties in implementing more than six identical shRNAs into a single plasmid.
T134 50208-50368 Epistemic_statement denotes However, the resulting disproportional expression levels (based on promoter strength) might inadvertently obscure the contribution of the individual components.
T135 50369-50538 Epistemic_statement denotes Alternatively, as already demonstrated and perhaps preferred, vectors can be engineered to express multiple miRNA-like hairpins from a single RNA polymerase II promoter.
T136 50620-50876 Epistemic_statement denotes On the other hand, the discrepant findings available on the efficiency of multi-miRNA vectors clearly indicate that implementation of this strategy requires an improved understanding of the cellular mechanisms that govern processing of hairpin concatemers.
T137 50877-51042 Epistemic_statement denotes As with any novel therapy, a stringent test for coRNAi strategies will be their evaluation in animal models of innate or acquired genetic disease or viral infection.
T138 51043-51260 Epistemic_statement denotes Importantly, in vivo trials will not only allow us to evaluate the efficacy of the new vectors directly but also provide us with better clues on the physiological role of the putative virally encoded RNAi suppressors.
T139 51261-51543 Epistemic_statement denotes Thus far, the majority of related findings have been obtained in artificial systems, using either robust plasmids for SRS expression (as opposed to perhaps low-level expression from the intact virus) or heterologous read-outs (e.g., using nonvertebrate cells for mammalian factors).
T140 51890-52142 Epistemic_statement denotes One example is HCV, and as we are now fortunate to have the first replication-competent wild-type isolate available, it will be possible and exciting to study the seemingly intricate interplay of the virus with the RNAi machinery in a natural scenario.
T141 52143-52283 Epistemic_statement denotes 1 The lessons learned will certainly influence the future design of coRNAi vectors with respect to the importance of SRS-specific silencers.
T142 52912-53137 Epistemic_statement denotes In conclusion, we anticipate with excitement the elucidation of whether coRNAi technology will live up to its promise in clinical studies and ultimately prove to be our winning strategy in the battle against evolving targets.
T143 53549-53716 Epistemic_statement denotes Particularly exciting candidates emerging as potential future RNAi partners are aptamers, RNA oligonucleotides able to bind ligands with high specificity and affinity.
T144 53717-53934 Epistemic_statement denotes In fact, recent work demonstrates that RNA aptamers can be expressed from RNA polymerase III promoters, identical to shRNAs, opening up the possibility of combining them with RNAi triggers in a multi-component vector.
T145 53935-54082 Epistemic_statement denotes 83 Moreover, aptamers can be fused with siRNAs to permit targeted RNAi delivery 84 or can be incorporated into shRNA loops as a regulatory element.
T146 54083-54343 Epistemic_statement denotes 81 Last but not least, by drawing upon our growing knowledge of endogenous RNAi pathways, the improvements in viral vector design, and the refinement of bioinformatical models of viral infection, we will be able further to enhance the efficacy of the approach.