1. Nagendra P2. Pramod M Jadhav

Short Hairpin RNA

RNA functions• Storage/transfer of genetic information

• Structural

• Catalytic

• Regulatory

RNAi• RNA interference (RNAi) is a biological process in which RNA molecules

inhibit gene expression, by the destruction of specific mRNA molecules.

• Historically, it was known by other names, includingco-suppression, post-transcriptional gene silencing (PTGS), and quelling.

• Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm Caenorhabditis elegans.

• RNAi is now known as precise, efficient, stable and better than antisense technology for gene suppression.

• microRNA (miRNA) and • small interfering RNA (siRNA) – are central to RNA interference.

C. elegans

Antisense Technology

Sh RNA• Small hairpin RNA or short hairpin RNA (shRNA)

is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference(RNAi).

• Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors.

• shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover.

Construction of shRNA

• The promoter choice is essential to achieve robust shRNA expression. At first, polymerase III promoters such as U6 ((It is believed that mouse U6 promoter transcription starts at the +1 position (23 nt after the TATA box), with G as the preferred initiation nucleotide)) and H1 were used; however, these promoters lack spatial and temporal control. As such, there has been a shift to using polymerase II promoters to regulate shRNA expression.

ApplicationAgriculture• Improving Disease Resistance• Improving Insect and Nematode Resistance• Improving Resistance against Parasitic Weeds• Improving Drought Tolerance• Improving Nutritional Value

Medicine• Gradalis, Inc. developed the FANG vaccine, which

is used in treatment of advanced cancers.• Marina Biotech developed CEQ508 which is used to

treat Familial Adenomatous Polyposis.

ChallengesSeveral challenges typically confront shRNA-based therapeutics.

• viral based gene therapy approaches have proved dangerous in past clinical trials. Some patients treated with viral vectors for Wiskott-Aldrich syndrome developed acute T-cell leukaemia.

• If the shRNA is expressed at levels that are too high the cell might not be able to correctly process the endogenous RNA which could cause significant problems.

• Another challenge is the possibility that the patient might mount an immune response against the therapy.

• Finally, there might be off-target effects and the shRNA could silence other unintended genes.

References• Silhavy, D. (2005) Agro-infiltration: A versatile tool for RNAi studies in

plants, in Gene silencing by RNA interference: Technology and application (Sohail, M., Ed.), pp 357-363, CRC press, Boca Raton.

• Paddison, PJ; Caudy, AA; Bernstein, E; Hannon, GJ; Conklin, DS (15 April 2002). “Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.”. Genes & Development. 16 (8): 948–58. doi:10.1101/gad.981002. PMID 11959843.

• Burnett, John C.; Rossi, John J.; Tiemann, Katrin (2011). “Current progress of siRNA/shRNA therapeutics in clinical trials”. Biotechnology Journal. 6 (9): 1130–1146. doi:10.1002/biot.201100054. ISSN 1860-6768.

• Bagasra O, Prilliman KR (2004). “RNA interference: the molecular immune system” (PDF). J. Mol. Histol. 35 (6): 545–53. doi:10.1007/s10735-004-2192-8. PMID 15614608.

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