Editing Small interfering RNA (section)

==Challenges: avoiding nonspecific effects==
RNAi intersects with a number of other pathways; as of 2010 it was not surprising that on occasion, nonspecific effects are triggered by the experimental introduction of an siRNA.<ref>{{cite journal | vauthors = Jackson AL, Linsley PS | title = Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application | journal = Nature Reviews Drug Discovery | volume = 9 | issue = 1 | pages = 57–67 | date = January 2010 | pmid = 20043028 | doi = 10.1038/nrd3010 | s2cid = 20903257 }}</ref><ref name=pmid1380154>{{cite journal | vauthors = Woolf TM, Melton DA, Jennings CG | title = Specificity of antisense oligonucleotides in vivo | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 89 | issue = 16 | pages = 7305–9 | date = August 1992 | pmid = 1380154 | pmc = 49698 | doi = 10.1073/pnas.89.16.7305 | bibcode = 1992PNAS...89.7305W | doi-access = free }}</ref> When a mammalian cell encounters a double-stranded RNA such as an siRNA, it may mistake it as a viral by-product and mount an immune response.  Furthermore, because structurally related [[microRNA]]s modulate gene expression largely via incomplete complementarity base pair interactions with a target [[Messenger RNA|mRNA]], the introduction of an siRNA may cause unintended off-targeting. Chemical modifications of siRNA may alter the thermodynamic properties that also result in a loss of single nucleotide specificity.<ref>{{cite journal | vauthors = Dua P, Yoo JW, Kim S, Lee DK | title = Modified siRNA structure with a single nucleotide bulge overcomes conventional siRNA-mediated off-target silencing | journal = Molecular Therapy | volume = 19 | issue = 9 | pages = 1676–87 | date = September 2011 | pmid = 21673662 | pmc = 3182346 | doi = 10.1038/mt.2011.109 }}</ref>

===Innate immunity===
Introduction of too many siRNA can result in nonspecific events due to activation of innate immune responses.<ref name=pmid22432611>{{cite journal | vauthors = Whitehead KA, Dahlman JE, Langer RS, Anderson DG | title = Silencing or stimulation? siRNA delivery and the immune system | journal = Annual Review of Chemical and Biomolecular Engineering | volume = 2 | issue = 1 | pages = 77–96 | date = 17 June 2011 | pmid = 22432611 | doi = 10.1146/annurev-chembioeng-061010-114133 | s2cid = 28803811 }}</ref> Most evidence to date suggests that this is probably due to activation of the dsRNA sensor PKR, although retinoic acid-inducible gene I (RIG-I) may also be involved.<ref>{{cite journal | vauthors = Matsumiya T, Stafforini DM | title = Function and regulation of retinoic acid-inducible gene-I | journal = Critical Reviews in Immunology | volume = 30 | issue = 6 | pages = 489–513 | date = 2010 | pmid = 21175414 | pmc = 3099591 | doi = 10.1615/critrevimmunol.v30.i6.10 }}</ref> The induction of cytokines via toll-like receptor 7 (TLR7) has also been described. Chemical modification of siRNA is employed to reduce in the activation of the innate immune response for gene function and therapeutic applications.  One promising method of reducing the nonspecific effects is to convert the siRNA into a microRNA.<ref>{{cite journal | vauthors = Barøy T, Sørensen K, Lindeberg MM, Frengen E | title = shRNA expression constructs designed directly from siRNA oligonucleotide sequences | journal = Molecular Biotechnology | volume = 45 | issue = 2 | pages = 116–20 | date = June 2010 | pmid = 20119685 | doi = 10.1007/s12033-010-9247-8 | s2cid = 24309609 }}</ref> MicroRNAs occur naturally, and by harnessing this endogenous pathway it should be possible to achieve similar gene knockdown at comparatively low concentrations of resulting siRNAs. This should minimize nonspecific effects.

===Off-targeting ===
Off-targeting is another challenge to the use of siRNAs as a gene knockdown tool.<ref name=pmid1380154/> Here, genes with incomplete complementarity are inadvertently downregulated by the siRNA (in effect, the siRNA acts as a miRNA), leading to problems in data interpretation and potential toxicity. This, however, can be partly addressed by designing appropriate control experiments, and siRNA design algorithms are currently being developed to produce siRNAs free from off-targeting. Genome-wide expression analysis, e.g., by microarray technology, can then be used to verify this and further refine the algorithms. A 2006 paper from the laboratory of Dr. Khvorova implicates 6- or 7-basepair-long stretches from position 2 onward in the siRNA matching with 3'UTR regions in off-targeted genes.<ref>{{cite journal | vauthors = Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y, Baskerville S, Maksimova E, Robinson K, Karpilow J, Marshall WS, Khvorova A | display-authors = 6 | title = 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets | journal = Nature Methods | volume = 3 | issue = 3 | pages = 199–204 | date = March 2006 | pmid = 16489337 | doi = 10.1038/nmeth854 | s2cid = 52809577 }}</ref> The tool of siRNA off-target predition is available at http://crdd.osdd.net/servers/aspsirna/asptar.php and published as ASPsiRNA resource.<ref name="pmid286969212">{{cite journal|vauthors=Monga I, Qureshi A, Thakur N, Gupta AK, Kumar M|year=2017|title=ASPsiRNA: A Resource of ASP-siRNAs Having Therapeutic Potential for Human Genetic Disorders and Algorithm for Prediction of Their Inhibitory Efficacy|url=http://crdd.osdd.net/servers/aspsirna/asptar.php|journal=[[G3: Genes, Genomes, Genetics ]]|volume=7|issue=9|pages=2931–2943|doi=10.1534/g3.117.044024|pmc=5592921|pmid=28696921}}</ref>

=== Adaptive immune responses ===
Plain RNAs may be poor immunogens, but antibodies can easily be created against RNA-protein complexes. Many autoimmune diseases see these types of antibodies. There haven't yet been reports of antibodies against siRNA bound to proteins. Some methods for siRNA delivery adjoin polyethylene glycol (PEG) to the oligonucleotide reducing excretion and improving circulating half-life. However recently a large Phase III trial of PEGylated RNA aptamer against factor IX had to be discontinued by Regado Biosciences because of a severe anaphylactic reaction to the PEG part of the RNA. This reaction led to death in some cases and raises significant concerns about siRNA delivery when PEGylated oligonucleotides are involved.<ref>{{cite journal | vauthors = Wittrup A, Lieberman J | title = Knocking down disease: a progress report on siRNA therapeutics | journal = Nature Reviews. Genetics | volume = 16 | issue = 9 | pages = 543–52 | date = September 2015 | pmid = 26281785 | pmc = 4756474 | doi = 10.1038/nrg3978 }}</ref>

=== Saturation of the RNAi machinery ===
siRNAs transfection into cells typically lowers the expression of many genes, however, the upregulation of genes is also observed. The upregulation of gene expression can partially be explained by the predicted gene targets of endogenous miRNAs. Computational analyses of more than 150 siRNA transfection experiments support a model where exogenous siRNAs can saturate the endogenous RNAi machinery, resulting in the de-repression of endogenous miRNA-regulated genes.<ref>{{cite journal | vauthors = Khan AA, Betel D, Miller ML, Sander C, Leslie CS, Marks DS | title = Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs | journal = Nature Biotechnology | volume = 27 | issue = 6 | pages = 549–55 | date = June 2009 | pmid = 19465925 | pmc = 2782465 | doi = 10.1038/nbt.1543 }}</ref> Thus, while siRNAs can produce unwanted off-target effects, i.e. unintended downregulation of mRNAs via a partial sequence match between the siRNA and target, the saturation of RNAi machinery is another distinct nonspecific effect, which involves the de-repression of miRNA-regulated genes and results in similar problems in data interpretation and potential toxicity.<ref>{{cite journal | vauthors = Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA | display-authors = 6 | title = Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways | journal = Nature | volume = 441 | issue = 7092 | pages = 537–41 | date = May 2006 | pmid = 16724069 | doi = 10.1038/nature04791 | s2cid = 15118504 | bibcode = 2006Natur.441..537G }}</ref>