Editing Small interfering RNA (section)

==Therapeutic applications and challenges==
Given the ability to knock down, in essence, any gene of interest, [[RNA interference|RNAi]] via siRNAs has generated a great deal of interest in both basic<ref>{{cite journal | vauthors = Alekseev OM, Richardson RT, Alekseev O, O'Rand MG | title = Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP | journal = Reproductive Biology and Endocrinology | volume = 7 | issue = 1 | pages = 45 | date = May 2009 | pmid = 19439102 | pmc = 2686705 | doi = 10.1186/1477-7827-7-45 | doi-access = free }}</ref> and applied biology.<ref>{{Cite journal | vauthors = Mahfuz A, Khan MA, Sajib EH, Deb A, Mahmud S, Hasan M, Saha O, Islam A, Rahaman MM |title=Designing potential siRNA molecules for silencing the gene of the nucleocapsid protein of Nipah virus: A computational investigation |journal=Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases |volume=102 |pages=105310 | date = August 2022 |pmid=35636695 |doi=10.1016/j.meegid.2022.105310 |doi-access = free |bibcode=2022InfGE.10205310M |issn=1567-7257}}</ref>

One of the biggest challenges to siRNA and RNAi based therapeutics is intracellular delivery.<ref name = "Petrocca_2011">{{cite journal | vauthors = Petrocca F, Lieberman J | title = Promise and challenge of RNA interference-based therapy for cancer | journal = Journal of Clinical Oncology | volume = 29 | issue = 6 | pages = 747–54 | date = February 2011 | pmid = 21079135 | doi = 10.1200/JCO.2009.27.6287 | s2cid = 15337692 }}</ref> siRNA also has weak stability and [[Pharmacokinetics|pharmacokinetic]] behavior.<ref name = "Hu_2020">{{cite journal | vauthors = Hu B, Zhong L, Weng Y, Peng L, Huang Y, Zhao Y, Liang XJ | title = Therapeutic siRNA: state of the art | journal = Signal Transduction and Targeted Therapy | volume = 5 | issue = 1 | pages = 101 | date = June 2020 | pmid = 32561705 | pmc = 7305320 | doi = 10.1038/s41392-020-0207-x }}</ref> Delivery of siRNA via [[Nanoparticle–biomolecule conjugate|nanoparticles]] has shown promise.<ref name = "Petrocca_2011" /> siRNA [[Oligonucleotide|oligos]] in vivo are vulnerable to degradation by plasma and tissue  [[nuclease|endonucleases and exonuclease]]s<ref name="Shen_2012">{{cite journal | vauthors = Shen H, Sun T, Ferrari M | title = Nanovector delivery of siRNA for cancer therapy | journal = Cancer Gene Therapy | volume = 19 | issue = 6 | pages = 367–73 | date = June 2012 | pmid = 22555511 | pmc = 3842228 | doi = 10.1038/cgt.2012.22 }}</ref> and have shown only mild effectiveness in localized delivery sites, such as the human eye.<ref name = "Burnett_2012">{{cite journal | vauthors = Burnett JC, Rossi JJ | title = RNA-based therapeutics: current progress and future prospects | journal = Chemistry & Biology | volume = 19 | issue = 1 | pages = 60–71 | date = January 2012 | pmid = 22284355 | pmc = 3269031 | doi = 10.1016/j.chembiol.2011.12.008 }}</ref> Delivering pure DNA to target organisms is challenging because its large size and structure prevents it from diffusing readily across [[membrane]]s.<ref name = "Petrocca_2011" /> siRNA oligos circumvent this problem due to their small size of 21-23 oligos.<ref name=pmid11157775>{{cite journal | vauthors = Elbashir SM, Lendeckel W, Tuschl T | title = RNA interference is mediated by 21- and 22-nucleotide RNAs | journal = Genes & Development | volume = 15 | issue = 2 | pages = 188–200 | date = January 2001 | pmid = 11157775 | pmc = 312613 | doi = 10.1101/gad.862301 }}</ref> This allows delivery via nano-scale delivery vehicles called nanovectors.<ref name = "Burnett_2012" />

A good nanovector for siRNA delivery should protect siRNA from degradation, enrich siRNA in the target organ and facilitate the cellular uptake of siRNA.<ref name="Shen_2012"/> The three main groups of siRNA nanovectors are: lipid based, non-lipid organic-based, and inorganic.<ref name="Shen_2012"/> [[Lipid]] based nanovectors are excellent for delivering siRNA to solid tumors,<ref name="Shen_2012"/> but other cancers may require different non-lipid based organic nanovectors such as [[cyclodextrin]] based nanoparticles.<ref name="Shen_2012"/><ref name=pmid17379663>{{cite journal | vauthors = Heidel JD, Yu Z, Liu JY, Rele SM, Liang Y, Zeidan RK, Kornbrust DJ, Davis ME | display-authors = 6 | title = Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 104 | issue = 14 | pages = 5715–21 | date = April 2007 | pmid = 17379663 | pmc = 1829492 | doi = 10.1073/pnas.0701458104 | doi-access = free | bibcode = 2007PNAS..104.5715H }}</ref>

siRNAs delivered via lipid based nanoparticles have been shown to have therapeutic potential for [[central nervous system]] ([[CNS disorders|CNS) disorders]].<ref name="Gomes et al 2016">{{cite journal |last1=Gomes |first1=Maria João |last2=Dreier |first2=Jes |last3=Brewer |first3=Jonathan |last4=Martins |first4=Susana |last5=Brandl |first5=Martin |last6=Sarmento |first6=Bruno | name-list-style = vanc |title=A new approach for a blood-brain barrier model based on phospholipid vesicles: Membrane development and siRNA-loaded nanoparticles permeability |journal=Journal of Membrane Science |date=April 2016 |volume=503 |pages=8–15 |doi=10.1016/j.memsci.2016.01.002 }}</ref> Central nervous disorders are not uncommon, but the [[Blood–brain barrier|blood brain barrier]] (BBB) often blocks access of potential therapeutics to the [[brain]].<ref name="Gomes et al 2016"/> siRNAs that target and silence efflux proteins on the BBB surface have been shown to create an increase in BBB permeability.<ref name="Gomes et al 2016"/> siRNA delivered via lipid based nanoparticles is able to cross the BBB completely.<ref name="Gomes et al 2016"/>

A huge difficulty in siRNA delivery is the problem of off-targeting.<ref name = "Petrocca_2011" /><ref name = "Burnett_2012" /> Since genes are read in both directions, there exists a possibility that even if the intended antisense siRNA strand is read and knocks out the target mRNA, the sense siRNA strand may target another protein involved in another function.<ref name=pmid25157701>{{cite journal | vauthors = Shukla RS, Qin B, Cheng K | title = Peptides used in the delivery of small noncoding RNA | journal = Molecular Pharmaceutics | volume = 11 | issue = 10 | pages = 3395–408 | date = October 2014 | pmid = 25157701 | pmc = 4186677 | doi = 10.1021/mp500426r }}</ref>

Phase I results of the first two therapeutic RNAi trials (indicated for [[age-related macular degeneration]], aka AMD) reported at the end of 2005 that siRNAs are well tolerated and have suitable pharmacokinetic properties.<ref>{{cite news |last1=Tansey |first1=Bernadette | name-list-style = vanc |title=Promising eye drug from S.F. firm / Macular degeneration treatment interferes with RNA messages |url=https://www.sfgate.com/business/article/Promising-eye-drug-from-S-F-firm-Macular-2514226.php |work=SFGATE |date=11 August 2006 }}</ref>

In a phase 1 clinical trial, 41 patients with advanced cancer [[metastasis]]ed to liver were [[RNAi nanoparticles to target cancer|administered RNAi]] delivered through [[nanomedicine#Drug delivery|lipid nanoparticles]]. The RNAi targeted two genes encoding key proteins in the growth of the cancer cells, vascular endothelial growth factor, ([[VEGF]]), and kinesin spindle protein ([[kinesin#kinesin and mitosis|KSP]]).  The results showed clinical benefits, with the cancer either stabilized after six months, or regression of metastasis in some of the patients.  [[Pharmacodynamic]] analysis of [[biopsy]] samples from the patients revealed the presence of the RNAi constructs in the samples, proving that the molecules reached the intended target.<ref>{{cite press release |title=First-in-man study demonstrates the therapeutic effect of RNAi gene silencing in cancer treatment |publisher=Vall d'Hebron Institute of Oncology |date=11 February 2013 |url=https://www.eurekalert.org/pub_releases/2013-02/vdio-fsd021113.php }}</ref><ref>{{cite journal | vauthors = Tabernero J, Shapiro GI, LoRusso PM, Cervantes A, Schwartz GK, Weiss GJ, Paz-Ares L, Cho DC, Infante JR, Alsina M, Gounder MM, Falzone R, Harrop J, White AC, Toudjarska I, Bumcrot D, Meyers RE, Hinkle G, Svrzikapa N, Hutabarat RM, Clausen VA, Cehelsky J, Nochur SV, Gamba-Vitalo C, Vaishnaw AK, Sah DW, Gollob JA, Burris HA | display-authors = 6 | title = First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement | journal = Cancer Discovery | volume = 3 | issue = 4 | pages = 406–17 | date = April 2013 | pmid = 23358650 | doi = 10.1158/2159-8290.CD-12-0429 | doi-access = free }}</ref>

Proof of concept trials have indicated that Ebola-targeted siRNAs may be effective as post-exposure prophylaxis in humans, with 100% of non-human primates surviving a lethal dose of Zaire Ebolavirus, the most lethal strain.<ref name=pmid20511019>{{cite journal | vauthors = Geisbert TW, Lee AC, Robbins M, Geisbert JB, Honko AN, Sood V, Johnson JC, de Jong S, Tavakoli I, Judge A, Hensley LE, Maclachlan I | display-authors = 6 | title = Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study | journal = Lancet | volume = 375 | issue = 9729 | pages = 1896–905 | date = May 2010 | pmid = 20511019 | pmc = 7138079 | doi = 10.1016/S0140-6736(10)60357-1 }}</ref>

=== Legal categorization and legal issues in a near future ===
Currently, SiRNA are currently chemically synthesized and so, are legally categorized inside EU and in USA as simple medicinal products. But as bioengineered siRNA (BERAs) are in development, these would be classified as biological medicinal products, at least in EU. The development of the BERAs technology raises the question of the categorization of drugs having the same mechanism of action but being produced chemically or biologically. This lack of consistency should be addressed.<ref>{{Cite journal |last1=Guerriaud |first1=Mathieu |last2=Kohli |first2=Evelyne |date=2022 |title=RNA-based drugs and regulation: Toward a necessary evolution of the definitions issued from the European union legislation |journal=Frontiers in Medicine |volume=9 |doi=10.3389/fmed.2022.1012497 |pmid=36325384 |pmc=9618588 |issn=2296-858X|doi-access=free }}</ref>