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• W2014018936 abstract "RNA interference has become a ubiquitous biological tool, and is being harnessed for therapeutic purposes as well. Therapeutic posttranscriptional gene silencing takes advantage of the endogenous RNAi pathway through delivery of either chemically synthesized siRNAs, or transgenes expressing hairpin-based inhibitory RNAs (e.g., shRNAs and artificial miRNAs). RNAi has expanded the field of viral gene therapy from gene replacement to gene knockdown. Here, we review various noncoding RNAs such as shRNAs, miRNAs, and miRNA decoys which can be utilized for therapeutic applications when expressed from recombinant adeno-associated vectors (AAV), and present examples of their basic design. In addition the basis of exploiting cellular miRNA profiles for detargeting AAV expression from specific cells is described. Finally, an overview of AAV-mediated RNAi preclinical studies is presented, and current RNAi-based clinical trials are reviewed. RNA interference has become a ubiquitous biological tool, and is being harnessed for therapeutic purposes as well. Therapeutic posttranscriptional gene silencing takes advantage of the endogenous RNAi pathway through delivery of either chemically synthesized siRNAs, or transgenes expressing hairpin-based inhibitory RNAs (e.g., shRNAs and artificial miRNAs). RNAi has expanded the field of viral gene therapy from gene replacement to gene knockdown. Here, we review various noncoding RNAs such as shRNAs, miRNAs, and miRNA decoys which can be utilized for therapeutic applications when expressed from recombinant adeno-associated vectors (AAV), and present examples of their basic design. In addition the basis of exploiting cellular miRNA profiles for detargeting AAV expression from specific cells is described. Finally, an overview of AAV-mediated RNAi preclinical studies is presented, and current RNAi-based clinical trials are reviewed. Since the first reports of the phenomenon in Petunia hybrida L. in the 1990's,1Napoli C Lemieux C Jorgensen R Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans.Plant Cell. 1990; 2: 279-289Crossref PubMed Google Scholar,2van der Krol AR Mur LA Beld M Mol JN Stuitje AR Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression.Plant Cell. 1990; 2: 291-299Crossref PubMed Scopus (884) Google Scholar its description in C. elegans in 1998,3Fire A Xu S Montgomery MK Kostas SA Driver SE Mello CC Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature. 1998; 391: 806-811Crossref PubMed Scopus (11739) Google Scholar and its identification in mammalian cells in 20014Elbashir SM Harborth J Lendeckel W Yalcin A Weber K Tuschl T Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature. 2001; 411: 494-498Crossref PubMed Scopus (8144) Google Scholar and mice in 2002,5McCaffrey AP Meuse L Pham TT Conklin DS Hannon GJ Kay MA RNA interference in adult mice.Nature. 2002; 418: 38-39Crossref PubMed Scopus (972) Google Scholar RNA interference, or RNAi, has sustained the interest of the scientific community. Not only does it offer a powerful biological tool, but also new therapeutic possibilities for nondruggable targets. Similarly, RNAi expanded the field of viral gene therapy from gene replacement to gene knockdown, quickly replacing other strategies such as ribozymes and antisense transcripts. Importantly, it has also opened up the field to tackle genetic disorders that result from both a loss-of-function and toxic gain-of-function.6Mao H Gorbatyuk MS Rossmiller B Hauswirth WW Lewin AS Long-term rescue of retinal structure and function by rhodopsin RNA replacement with a single adeno-associated viral vector in P23H RHO transgenic mice.Hum Gene Ther. 2012; 23: 356-366Crossref PubMed Scopus (64) Google Scholar,7Mueller C Tang Q Gruntman A Blomenkamp K Teckman J Song L et al.Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.Mol Ther. 2012; 20: 590-600Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Since the awarding of a 2006 Nobel Prize for the description of RNAi, academic groups as well as the pharmaceutical industry have shown interest in its application. This has grown into a reported 162 companies involved in the development of RNAi technologies, with 33 developing RNAi therapeutics and 35 developing microRNA (miRNA) therapeutics.8Jain KK RNAi: Technologies, markets and companies. Jain PharmaBiotech, Basel, Switzerland2013Google Scholar RNAi resulting from the endogenous miRNA pathway regulates gene expression by controlling the synthesis of protein through posttranscriptional gene silencing.9Lee RC Feinbaum RL Ambros V The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell. 1993; 75: 843-854Abstract Full Text PDF PubMed Scopus (9648) Google Scholar The miRNA biogenesis will only be briefly described here; for more details on this topic refer to a recent review.10Ameres SL Zamore PD Diversifying microRNA sequence and function.Nat Rev Mol Cell Biol. 2013; 14: 475-488Crossref PubMed Scopus (900) Google Scholar Aside from a few exceptions,11Borchert GM Lanier W Davidson BL RNA polymerase III transcribes human microRNAs.Nat Struct Mol Biol. 2006; 13: 1097-1101Crossref PubMed Scopus (1042) Google Scholar,12Canella D Praz V Reina JH Cousin P Hernandez N Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells.Genome Res. 2010; 20: 710-721Crossref PubMed Scopus (132) Google Scholar the miRNA gene is usually transcribed by RNA polymerase II (RNA Pol II) from independent transcription units or from the intron of protein coding genes into a ∼1–4 kb13Saini HK Griffiths-Jones S Enright AJ Genomic analysis of human microRNA transcripts.Proc Natl Acad Sci USA. 2007; 104: 17719-17724Crossref PubMed Scopus (409) Google Scholar primary transcript called a pri-miRNA (Figure 1). In mammals the pri-miRNA is cleaved by the microprocessor complex consisting of RNAse III Drosha and the RNA-binding protein DiGeorge syndrome critical region gene 8 (DGRC8) into a ∼60–80 nt-long precursor miRNA (pre-miRNA). The pre-miRNA di-nucleotide 3′ overhang and stem-loop get recognized by Exportin 5 which exports it from the nucleus to the cytoplasm via the nuclear pore (Figure 1). In the cytoplasm, the pre-miRNA is bound by a second RNAse III enzyme, known as Dicer. Much like the microprocessor complex, Dicer and its partner proteins, protein kinase R activator (PACT) and TAR RNA-binding protein (TRBP), recognize the base of the hairpin stem and cleave at a fixed distance. This measured interaction results in cleavage of the loop and the production of ∼22nt-long miRNA-miRNA* duplex (Figure 1). Importantly, at this point in the pathway, RNAi can also be triggered by exogenous double-stranded RNA (dsRNA) which can be processed by Dicer to give a ∼22 nt-long siRNA-siRNA*duplex. After pre-miRNA processing, the mature miRNA or functional siRNA or miRNA* strand (i.e., the antisense guide strand which is complementary to the messenger RNA (mRNA)) is incorporated into the “RNA-induced silencing complex” (RISC), while the opposite strand (i.e., the passenger strand) is degraded. At the core of the catalytic domain of the RISC are the Ago proteins. Ago proteins consist of four distinct domains: the N-terminal, PAZ, Mid, and PIWI domains. The PIWI domain resembles a RNase H-like enzyme. In some Ago family members (e.g., Ago2) this domain retains a functional catalytic center allowing for the cleavage of a target mRNA (Figure 1). The seed sequence (6–8 nt of the 5′ end) of the guide strand leads the RISC to bind to the mRNA by sequence complementarity. This leads to translational repression, enhanced mRNA degradation or site-specific mRNA cleavage. The later event is the most efficient inhibitory mechanism and only occurs if there is complete or near complete complementarity between the mRNA and the mi/siRNA. The earliest attempts to harness RNAi therapeutically were to deliver dsRNA molecules to feed into the RNAi pathway at the level of Dicer. For this approach, efficient delivery of RNAi effectors has been the greatest hurdle for clinical translation but there have been improvements in lipid-based siRNA delivery in the last few years.14Zhang Y Satterlee A Huang L In vivo gene delivery by nonviral vectors: overcoming hurdles?.Mol Ther. 2012; 20: 1298-1304Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar Another impediment has been that the direct administration of dsRNA in the form of siRNAs complexed to lipids has been shown to activate innate inflammatory pathways. Specifically, activation of the interferon response by the small RNA can trigger toxicity. Also, toll-like receptor 3 (TLR3), TLR7, and TLR8 can recognize siRNA in a sequence-dependent (TLR7 and TLR8) or -independent (TLR3) fashion, and induce interferon, interleukin 6, and tumor necrosis factor α.15Hornung V Guenthner-Biller M Bourquin C Ablasser A Schlee M Uematsu S et al.Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7.Nat Med. 2005; 11: 263-270Crossref PubMed Scopus (1051) Google Scholar,16Judge AD Sood V Shaw JR Fang D McClintock K MacLachlan I Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.Nat Biotechnol. 2005; 23: 457-462Crossref PubMed Scopus (997) Google Scholar,17Karikó K Bhuyan P Capodici J Weissman D Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3.J Immunol. 2004; 172: 6545-6549Crossref PubMed Scopus (388) Google Scholar Owing to the fact that siRNAs degrade over time, these responses can be limiting when one is faced with the notion of lifelong repeated administration in some cases. Fortunately, improved lipid chemistries, chemical modifications, and recent advances in eliminating specific motifs have reduced, but not abolished, these potential serious side-effects.14Zhang Y Satterlee A Huang L In vivo gene delivery by nonviral vectors: overcoming hurdles?.Mol Ther. 2012; 20: 1298-1304Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar Thus, the main limitations of siRNA therapeutics today are efficient delivery to organs other than the liver, longevity and the associated innate immune response with each dose. In cases where sustained expression of RNAi effectors is needed, a vector-derived approach such as the ones described below may be more adequate. Vector-derived RNAi is where a vector is used to express RNA transcripts (e.g., short-hairpin RNAs (shRNAs) or micro RNAs (miRNAs) that are ultimately processed to produce siRNAs in the target cells. In this review, we focus on the use of recombinant adeno-associated vectors (rAAV) to achieve this goal. Adeno-associated virus currently is one of the most attractive gene therapy vectors. First, the virus naturally infects primates and is nonpathogenic. Second, the recombinant form used for therapy, where the rep and cap genes have been removed, remains almost completely episomal with a reported 0.05% integration in neonatal mice18Inagaki K Piao C Kotchey NM Wu X Nakai H Frequency and spectrum of genomic integration of recombinant adeno-associated virus serotype 8 vector in neonatal mouse liver.J Virol. 2008; 82: 9513-9524Crossref PubMed Scopus (46) Google Scholar and between 10E−4 and 10E−5 in liver and muscle of nonhuman primates19Nowrouzi A Penaud-Budloo M Kaeppel C Appelt U Le Guiner C Moullier P et al.Integration frequency and intermolecular recombination of rAAV vectors in non-human primate skeletal muscle and liver.Mol Ther. 2012; 20: 1177-1186Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar and humans.20Kaeppel C Beattie SG Fronza R van Logtenstein R Salmon F Schmidt S et al.A largely random AAV integration profile after LPLD gene therapy.Nat Med. 2013; 19: 889-891Crossref PubMed Scopus (124) Google Scholar The relatively limited packaging capacity of AAV of 4.7 kb can be a disadvantage when designing vectors for gene replacement but not for RNAi-based applications, which typically employ smaller-sized expression cassettes. In recent times, rAAV vectors have continued to add to their safety profile by their evidence of therapeutic success in clinical trials (in particular for Leber's congenital amaurosis,21Cideciyan AV Hauswirth WW Aleman TS Kaushal S Schwartz SB Boye SL et al.Vision 1 year after gene therapy for Leber's congenital amaurosis.N Engl J Med. 2009; 361: 725-727Crossref PubMed Scopus (146) Google Scholar hemophilia B,22Nathwani AC Davidoff AM Hanawa H Hu Y Hoffer FA Nikanorov A et al.Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques.Blood. 2002; 100: 1662-1669Crossref PubMed Scopus (148) Google Scholar and lipoprotein lipase deficiency).23Gaudet D Méthot J Déry S Brisson D Essiembre C Tremblay G et al.Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial.Gene Ther. 2013; 20: 361-369Crossref PubMed Scopus (294) Google Scholar Below, we describe the use of AAV-based strategies for the expression of noncoding inhibitory RNAs as well as the use of noncoding RNAs to detarget AAV expression. We conclude with an overview of AAV-mediated RNAi preclinical studies, and current RNAi-based clinical trials. To date most of the AAV-RNAi approaches takes either the form of a shRNA or pri-miRNA as the effector molecule. While the design differences are subtle, and they both converge to silence targets via the RISC, they are processed differently, which as described below, may have important consequences. For guidance on how to design and clone either of these into AAV vectors refer to the following protocol.24Boudreau RL Davidson BL Generation of hairpin-based RNAi vectors for biological and therapeutic application.Meth Enzymol. 2012; 507: 275-296Crossref PubMed Scopus (34) Google Scholar ShRNAs are stem-loop RNAs that bypass Drosha processing and are incorporated in the pathway directly at the point of Exportin 5 followed by Dicer cleavage (Figure 1) to generate siRNAs. An example of a shRNA with its typical structure is presented in Figure 2a. shRNAs have been widely used for gene knockdown applications because of their simple design and, when expressed from an AAV vector for their long-term expression and stability. However, in 2006, concerns about toxicity of shRNA molecules were raised by the report of severe liver injury, and in some cases death, in mice injected with high dose AAV8-shRNAs.25Grimm D Streetz KL Jopling CL Storm TA Pandey K Davis CR et al.Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.Nature. 2006; 441: 537-541Crossref PubMed Scopus (1372) Google Scholar The hepatocellular toxicity appeared to be sequence-independent as it was observed with 36 out of 49 tested shRNAs, ultimately resulting in morbidity within 2 months with 23 different shRNA constructs. At the time it was hypothesized that high levels of shRNAs competed with endogenous miRNAs for intracellular processing and to the extent that it impeded vital cellular processes. This was partially corroborated by the fact that overexpression of Exportin 5, which exports pre-miRNAs (Figure 1), increased knockdown but also toxicity which suggested saturation of downstream factors as well.26Grimm D Wang L Lee JS Schürmann N Gu S Börner K et al.Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver.J Clin Invest. 2010; 120: 3106-3119Crossref PubMed Scopus (150) Google Scholar In fact, argonaute 2 was later shown to be another saturable factor in the RNAi pathway.26Grimm D Wang L Lee JS Schürmann N Gu S Börner K et al.Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver.J Clin Invest. 2010; 120: 3106-3119Crossref PubMed Scopus (150) Google Scholar,27Castanotto D Sakurai K Lingeman R Li H Shively L Aagaard L et al.Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC.Nucleic Acids Res. 2007; 35: 5154-5164Crossref PubMed Scopus (217) Google Scholar,28Diederichs S Jung S Rothenberg SM Smolen GA Mlody BG Haber DA Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites.Proc Natl Acad Sci USA. 2008; 105: 9284-9289Crossref PubMed Scopus (83) Google Scholar Many groups using AAV vectors to deliver RNAi constructs have since then reproducibly demonstrated that high levels of shRNAs, such as those generated by the classic RNA polymerase III promoters (RNA Pol III), will compete with the endogenous RNAi species, eventually leading to saturation of the cellular machinery.29Grimm D The dose can make the poison: lessons learned from adverse in vivo toxicities caused by RNAi overexpression.Silence. 2011; 2: 8Crossref PubMed Scopus (54) Google Scholar shRNAs are usually expressed from RNA Pol III promoters because of their well-defined initiation and termination sites; the most commonly used are the human U6 small nuclear RNA promoter and the human H1-RNA gene promoter,30Baer M Nilsen TW Costigan C Altman S Structure and transcription of a human gene for H1 RNA, the RNA component of human RNase P.Nucleic Acids Res. 1990; 18: 97-103Crossref PubMed Scopus (105) Google Scholar U6 being stronger than H1. Toxicity appears not to be U6-specific, as was evident from a study that used H1-driven shRNAs in the liver.31Borel F van Logtenstein R Koornneef A Maczuga P Ritsema T Petry H et al.In vivo knock-down of multidrug resistance transporters ABCC1 and ABCC2 by AAV-delivered shRNAs and by artificial miRNAs.J RNAi Gene Silencing. 2011; 7: 434-442PubMed Google Scholar In these studies with AAV-H1-shRNA, a viral dose-dependent hepatotoxicity was reported in mice, characterized by elevated transaminases levels, signs of stress and ultimately death, which appeared to be paralleled by a decrease in some endogenous miRNA levels, including miR-122.31Borel F van Logtenstein R Koornneef A Maczuga P Ritsema T Petry H et al.In vivo knock-down of multidrug resistance transporters ABCC1 and ABCC2 by AAV-delivered shRNAs and by artificial miRNAs.J RNAi Gene Silencing. 2011; 7: 434-442PubMed Google Scholar ShRNA toxicity is neither organ-specific nor species-specific. In the brain, Elhert et al. observed toxicities in the red nucleus leading to neuronal degeneration and showed that reducing the viral dose or using a less efficient serotype could alleviate this toxicity,32Ehlert EM Eggers R Niclou SP Verhaagen J Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system.BMC Neurosci. 2010; 11: 20Crossref PubMed Scopus (66) Google Scholar confirming the original work of Grimm et al.25Grimm D Streetz KL Jopling CL Storm TA Pandey K Davis CR et al.Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.Nature. 2006; 441: 537-541Crossref PubMed Scopus (1372) Google Scholar described above that first reported this toxicity. Cardiac toxicity was reported in dogs following injection of AAV-shRNA, which also appeared to be paralleled by a decrease in endogenous miRNA levels.33Bish LT Sleeper MM Reynolds C Gazzara J Withnall E Singletary GE et al.Cardiac gene transfer of short hairpin RNA directed against phospholamban effectively knocks down gene expression but causes cellular toxicity in canines.Hum Gene Ther. 2011; 22: 969-977Crossref PubMed Scopus (35) Google Scholar A majority of reported shRNA-associated toxicities occurred in the CNS, including the mouse striatum,34Martin JN Wolken N Brown T Dauer WT Ehrlich ME Gonzalez-Alegre P Lethal toxicity caused by expression of shRNA in the mouse striatum: implications for therapeutic design.Gene Ther. 2011; 18: 666-673Crossref PubMed Scopus (80) Google Scholar,35McBride JL Boudreau RL Harper SQ Staber PD Monteys AM Martins I et al.Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi.Proc Natl Acad Sci USA. 2008; 105: 5868-5873Crossref PubMed Scopus (477) Google Scholar mouse cerebellum,36Boudreau RL Martins I Davidson BL Artificial microRNAs as siRNA shuttles: improved safety as compared to shRNAs in vitro and in vivo.Mol Ther. 2009; 17: 169-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar rat substantia nigra37Khodr CE Sapru MK Pedapati J Han Y West NC Kells AP et al.An a-synuclein AAV gene silencing vector ameliorates a behavioral deficit in a rat model of Parkinson's disease, but displays toxicity in dopamine neurons.Brain Res. 2011; 1395: 94-107Crossref PubMed Scopus (86) Google Scholar,38Ulusoy A Sahin G Björklund T Aebischer P Kirik D Dose optimization for long-term rAAV-mediated RNA interference in the nigrostriatal projection neurons.Mol Ther. 2009; 17: 1574-1584Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar and rat red nucleus neurons.32Ehlert EM Eggers R Niclou SP Verhaagen J Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system.BMC Neurosci. 2010; 11: 20Crossref PubMed Scopus (66) Google Scholar In those studies, the authors observed neurotoxicity with several shRNAs including control shRNAs. In general, with shRNAs, there is a risk of high expression that has to be balanced with AAV serotype and promoter choice as well as vector dose. In summary, most of these studies indicate that supraphysiologic expression of these RNA species may be at the root of their toxicity. In fact, in some cases, switching shRNA expression from a RNA Pol III to a RNA Pol II promoter has been showed to alleviate toxicity, pointing to the fact that RNA Pol II promoters may be a safer approach for shRNA-mediated silencing.36Boudreau RL Martins I Davidson BL Artificial microRNAs as siRNA shuttles: improved safety as compared to shRNAs in vitro and in vivo.Mol Ther. 2009; 17: 169-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar,39Giering JC Grimm D Storm TA Kay MA Expression of shRNA from a tissue-specific pol II promoter is an effective and safe RNAi therapeutic.Mol Ther. 2008; 16: 1630-1636Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar Another potential source of toxicity is off-targeting, i.e., the downregulation of an undesired target containing a partial or complete homology to the si/miRNA being expressed. In fact unintended seed sequence homology as determined by a bioinformatics analysis has been shown to correlate with off-target transcriptional changes.40Anderson EM Birmingham A Baskerville S Reynolds A Maksimova E Leake D et al.Experimental validation of the importance of seed complement frequency to siRNA specificity.RNA. 2008; 14: 853-861Crossref PubMed Scopus (104) Google Scholar Off-targeting remains a major issue as it is difficult to predict and to identify. Moreover, preclinical studies are of limited use in this respect because off-targeting can be species-specific.41Burchard J Jackson AL Malkov V Needham RH Tan Y Bartz SR et al.MicroRNA-like off-target transcript regulation by siRNAs is species specific.RNA. 2009; 15: 308-315Crossref PubMed Scopus (71) Google Scholar Following rational design rules can help reduce off-targeting, in particular selecting artificial RNAi effectors whose seeds have a low incidence in 3′UTRs,42Boudreau RL Spengler RM Davidson BL Rational design of therapeutic siRNAs: minimizing off-targeting potential to improve the safety of RNAi therapy for Huntington's disease.Mol Ther. 2011; 19: 2169-2177Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar the predominant location of off-target events. Another cause of off-targeting is Dicer “slippage,” or noncanonical shRNA cleavage by Dicer, leading to different 5′ and 3′ cleavage sites and siRNA length,43Maczuga P Lubelski J van Logtenstein R Borel F Blits B Fakkert E et al.Embedding siRNA sequences targeting apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy.Mol Ther. 2013; 21: 217-227Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar which by creating a heterogeneous siRNA pool increases off-target multiplicity. It is not yet known if these “by-products” contribute to the toxicity. Nevertheless, to circumvent this, a new generation of shRNAs have been designed to have an internal 3-nt bulge, 2 nt away from the cleavage site of 29 bp-stem shRNAs which may be recognized by the Dicer helicase domain, and as a result imprecise cleavages are abrogated,44Gu S Jin L Zhang Y Huang Y Zhang F Valdmanis PN et al.The loop position of shRNAs and pre-miRNAs is critical for the accuracy of dicer processing in vivo.Cell. 2012; 151: 900-911Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar reducing off-targeting. Other methods to reduce shRNA toxicity involve bypassing Dicer all together. The 17–19 nt-long shRNAs named AgoshRNAs, which are shorter than the conventional 21 nt-long ones, appear to be directly incorporated into RISC and processed by AGO.45Liu YP Schopman NC Berkhout B Dicer-independent processing of short hairpin RNAs.Nucleic Acids Res. 2013; 41: 3723-3733Crossref PubMed Scopus (63) Google Scholar By avoiding imprecise Dicer cleavage, these shRNAs not only lead to more specific and more predictable siRNAs but also do not compete with this step of the pathway. Designing shRNA constructs which follow these additional rules will help prevent off-target effects and reduce toxicity. As mentioned above, another concept for reducing the extent of the off-targeting is using the lowest siRNA dose for sufficient knockdown of the desired target.46Jackson AL Burchard J Schelter J Chau BN Cleary M Lim L et al.Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity.RNA. 2006; 12: 1179-1187Crossref PubMed Scopus (738) Google Scholar Employing low concentrations of multiple siRNAs directed against the same target can maintain a sufficient knockdown while dissipating nonspecific effects, due to the differences between sequences.47Kittler R Surendranath V Heninger AK Slabicki M Theis M Putz G et al.Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies.Nat Methods. 2007; 4: 337-344Crossref PubMed Scopus (148) Google Scholar As mentioned earlier, AAV-RNAi is either achieved by delivery of a shRNA or an artificial miRNA. Early on, most of the studies centered on the use of shRNAs, probably due to their inherently simple design. As the field matured and some of above-mentioned evidence of shRNA-associated toxicity was reported, the use of artificial miRNAs (i.e., a cellular miRNA whose stem has been modified to be partially complementary to a mRNA of interest, Figure 2a) gained more popularity. Interestingly enough, many of the observed toxicities with shRNA have been alleviated by shifting to artificial miRNAs. However, it should be noted that using an artificial miRNA over a shRNA adds another potentially saturable step, the Drosha cleavage of the pri-miRNA into pre-miRNA (Figure 1), although to this date no such toxicity has been described. Like shRNAs, artificial miRNAs can be expressed from RNA Pol III but also RNA Pol II promoters, which have lower rate of expression but offer many possibilities in terms of tissue-specific and regulatable expression, all desirable options to avoid potential toxicity and off-target effects. The inherently reduced abundance and more efficient processing of artificial miRNAs over shRNAs48Boudreau RL Monteys AM Davidson BL Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs.RNA. 2008; 14: 1834-1844Crossref PubMed Scopus (112) Google Scholar may be due to the rate limiting cleavage of Drosha. Although this has not been formally proven, an elegant study points in that direction. The study used a fair comparison method in which the artificial miRNA and shRNA were both expressed from U6 promoters and were designed to yield the same siRNA after Dicer processing.36Boudreau RL Martins I Davidson BL Artificial microRNAs as siRNA shuttles: improved safety as compared to shRNAs in vitro and in vivo.Mol Ther. 2009; 17: 169-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar Thus, the only known difference was that the shRNA bypassed Drosha while the artificial miRNA was dependent on Drosha cleavage. Interestingly, the findings suggest that the shRNA disrupted endogenous miRNA biogenesis, whereas the designed artificial miRNA did not. This was attributed to the higher abundance of unprocessed shRNAs despite being expressed from the same promoter as the artificial miRNA.36Boudreau RL Martins I Davidson BL Artificial microRNAs as siRNA shuttles: improved safety as compared to shRNAs in vitro and in vivo.Mol Ther. 2009; 17: 169-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar Further testing in vivo showed that the shRNA-expressing vectors led to overt neurotoxicity in cerebellar Purkinje neurons.36Boudreau RL Martins I Davidson BL Artificial microRNAs as siRNA shuttles: improved safety as compared to shRNAs in vitro and in vivo.Mol Ther. 2009; 17: 169-175Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar The authors conclude that AAV-artificial miRNA vectors where just as efficient at silencing, but more importantly they resulted in red" @default.
• W2014018936 created "2016-06-24" @default.
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• W2014018936 date "2014-04-01" @default.
• W2014018936 modified "2023-10-18" @default.
• W2014018936 title "Recombinant AAV as a Platform for Translating the Therapeutic Potential of RNA Interference" @default.
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• W2014018936 doi "https://doi.org/10.1038/mt.2013.285" @default.
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