Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA.
Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.
Nature. 2006 May 25;441(7092):537-41.
Please login to recommend the paper.
Make a Comment
RNA interference (RNAi), a technique used to knock down gene and protein expression, is a relatively recent addition to the molecular biology toolkit. It has garnered extreme interest in all fields of biology because of its efficiency and ease of use. Indeed, it has been touted as the new therapy for a number of disorders, and clinical trials in humans are planned or underway. This is especially relevant for neurological disorders (see review by Bonini and LaSpada, 2005), and a number of recent papers have used RNAi in mouse models with some success, for example, in ALS (Ralph et al., 2005), Huntington disease (Harper et al., 2005 and see ARF related news story), and spinocerebellar ataxia (Xia et al., 2004 and see ARF related news story). Whether RNAi as a therapy is generally safe is still an open question. Early studies indicated that RNAi can induce a general interferon/immune reaction, although this has remained a controversial finding. There has also been some concern about off-target silencing effects. This paper by Mark Kay’s group sheds new light on the possible adverse side effects of RNAi therapy.
Grimm and colleagues delivered 49 different shRNAs (short hairpin RNAs) into mouse liver cells using viral vectors. They found that 36 of the shRNAs were severely toxic, and 23 killed the mice within two months. These results suggested that toxicity was not limited to specific RNAi targets—toxicity due to the viral vectors was also ruled out (although not shown). No interferon response was observed. The biological mechanism of precisely how RNAi works has been an intense area of research, and a number of important insights into normal cell biology have been discovered. One of these pertains to the role of microRNAs (miRNAs) in regulating cellular function. miRNAs have important roles in mRNA translation, cell cycle, and development. These cellular RNAs of about 22 nucleotides are processed by the same intracellular machinery as the shRNA constructs commonly used to induce RNAi. The authors found that the toxicity observed after administering the shRNAi was due to the saturation/competition of the intracellular processing machinery used to regulate miRNAs, leading to lower expression of these important regulatory molecules. The authors speculate that the key limiting component is exportin-5, the main function of which is the nuclear export and stabilization of shRNAs/miRNAs.
Do these findings damage the future use of RNAi in humans as putative therapies? Although certainly highlighting the great care that is needed in exploring a novel technique, this paper does not completely rule out RNAi as a therapy. These are some important points to consider: 1. Toxicity was only observed with extremely high levels of shRNAi expression, induced by high virus expression. 2. Low levels of shRNAi were nontoxic and still functional. 3. RNAi induced by direct application of 19-25mer oligos (siRNA) has not been reported to be toxic, and these oligomers do not compete with the intracellular miRNA processing machinery. 4. Some shRNAs were not toxic even when robustly expressed, suggesting that there may be a way of designing shRNAs that are safe, even at very high levels.
Though the study by Kay’s group is important because it delineates some crucial adverse effects induced by high levels of shRNA, it seems these effects can be avoided. With advances in siRNAi and inducible promoters for shRNA, the future of RNAi as a therapy is still full of potential.
Bonini NM, La Spada AR.
Silencing polyglutamine degeneration with RNAi.
Neuron. 2005 Dec 8;48(5):715-8.
Ralph GS, Radcliffe PA, Day DM, Carthy JM, Leroux MA, Lee DC, Wong LF, Bilsland LG, Greensmith L, Kingsman SM, Mitrophanous KA, Mazarakis ND, Azzouz M.
Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model.
Nat Med. 2005 Apr;11(4):429-33.
Harper SQ, Staber PD, He X, Eliason SL, Martins IH, Mao Q, Yang L, Kotin RM, Paulson HL, Davidson BL.
RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model.
Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5820-5.
Xia H, Mao Q, Eliason SL, Harper SQ, Martins IH, Orr HT, Paulson HL, Yang L, Kotin RM, Davidson BL.
RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia.
Nat Med. 2004 Aug;10(8):816-20.
To make a comment you must login or register.