Alvarez VA, Ridenour DA, Sabatini BL.
Retraction of synapses and dendritic spines induced by off-target effects of RNA interference.
J Neurosci. 2006 Jul 26;26(30):7820-5.
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Alvarez et al. show that some shRNAs, when expressed in cortical neurons, can cause RNAi-target-independent effects, including shortened dendrites, reduced spine density and length, and decreased synaptic transmission. Based on these results, they caution that studies using RNAi should include proper controls. Particularly important is a rescue experiment using the shRNA-resistant target construct.
This paper comes on the heels of several others raising a serious issue in designing RNAi experiments and therapeutic strategies. In general, there are three types of off-target effects in RNAi: off-target silencing; the interferon response; and the interference with the endogenous miRNA production.
Off-target silencing is the silencing of the unintended genes (Jackson and Linsley, 2004). An important contributor to this is homology in the 5’ half of the siRNA (called the seed sequence) with the sequences of the non-targeted mRNAs (Jackson and Linsley, 2004).
The interferon response is the expression and activation of antiviral genes (which include PKR as described by Alvarez et al.) by the cell after it is exposed to the double-stranded RNA. Many studies have contributed to the understanding of the mechanism whereby siRNA or shRNA triggers interferon response in mammalian cells. Among the contributing factors are the use of siRNA or shRNA synthesized by T7 polymerase (Kim et al., 2004; Sledz et al., 2003), the specific sequence motifs in the siRNA (Hornung et al., 2005; Judge et al., 2005), some characteristic of the shRNA expression vector (Bridge et al., 2003; Pebernard and Iggo, 2004), the inclusion or exclusion of the two 3’-overhang nucleotides (Marques et al., 2006), the length of the siRNA (Marques et al., 2006), the methods of siRNA or shRNA delivery (Heidel et al., 2004; Judge et al., 2005; Robbins et al., 2006), and even the purity of the chemically synthesized siRNAs (Robbins et al., 2006). Activation of the interferon response can trigger complex consequences that include apoptosis. Alvarez et al. showed that the off-target effects that they observed on neurons depend on the activity of PKR, suggesting that these effects are the consequence of the interferon response to neurons.
The interference with endogenous miRNA is caused by introducing a large amount of shRNA or siRNA into the cells. This can saturate the endogenous miRNA processing and effecting machinery, leading to reduced function of the endogenous miRNAs (Grimm et al., 2006). One manifestation of a compromised endogenous miRNA function is the reduction in the levels of endogenous miRNAs (Grimm et al., 2006). Another manifestation is the increased expression of target genes of the endogenous miRNA. Perhaps this explains why a large number of genes were reported to increase when high doses of siRNAs were transfected into cells (Persengiev et al., 2004). Based on studies on Dicer knockout mice and other studies on miRNA function in developing vertebrates (Song and Tuan, 2006), it is clear that the miRNA function is essential for the survival of mammals. Thus, interference of the endogenous miRNA function can cause serious cellular dysfunction and even death (Grimm et al., 2006).
With these multiple off-target effects, it is extremely important to carefully consider controls in reverse genetics experiments. Most useful in addressing these off-target effects are using multiple siRNA or shRNA to silence the target and conducting rescue experiments using the siRNA- or shRNA-resistant target constructs. A common character of these off-target effects is that they are siRNA or shRNA dose-dependent. Higher doses tend to trigger more severe off-target effects and can cause cell death. Therefore, the lowest effective silencing dose should always be used in reverse genetic experiments. Other helpful controls include measuring the expression levels of interferon response genes, checking the levels of endogenous miRNAs, and gene profiling of the RNAi treated cells.
As far as the therapeutic RNAi is concerned, this report raises new challenges. Therapeutic siRNA or shRNA should be screened and the ones with the lowest effective silencing dose and highest toxic dose should be selected and thoroughly tested for their off-target effects. Ideally, the dose of the therapeutic siRNA or shRNA should be controlled so that it will not exceed the non-toxic range.
Bridge AJ, Pebernard S, Ducraux A, Nicoulaz AL, Iggo R.
Induction of an interferon response by RNAi vectors in mammalian cells.
Nat Genet. 2003 Jul;34(3):263-4.
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.
Heidel JD, Hu S, Liu XF, Triche TJ, Davis ME.
Lack of interferon response in animals to naked siRNAs.
Nat Biotechnol. 2004 Dec;22(12):1579-82.
Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Schlee M, Uematsu S, Noronha A, Manoharan M, Akira S, de Fougerolles A, Endres S, Hartmann G.
Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7.
Nat Med. 2005 Mar;11(3):263-70.
Jackson AL, Linsley PS.
Noise amidst the silence: off-target effects of siRNAs?.
Trends Genet. 2004 Nov;20(11):521-4.
Judge 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 Apr;23(4):457-62.
Kim DH, Longo M, Han Y, Lundberg P, Cantin E, Rossi JJ.
Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase.
Nat Biotechnol. 2004 Mar;22(3):321-5.
Marques JT, Devosse T, Wang D, Zamanian-Daryoush M, Serbinowski P, Hartmann R, Fujita T, Behlke MA, Williams BR.
A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells.
Nat Biotechnol. 2006 May;24(5):559-65.
Pebernard S, Iggo RD.
Determinants of interferon-stimulated gene induction by RNAi vectors.
Differentiation. 2004 Mar;72(2-3):103-11.
Persengiev SP, Zhu X, Green MR.
Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs).
RNA. 2004 Jan;10(1):12-8.
Robbins MA, Li M, Leung I, Li H, Boyer DV, Song Y, Behlke MA, Rossi JJ.
Stable expression of shRNAs in human CD34+ progenitor cells can avoid induction of interferon responses to siRNAs in vitro.
Nat Biotechnol. 2006 May;24(5):566-71.
Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR.
Activation of the interferon system by short-interfering RNAs.
Nat Cell Biol. 2003 Sep;5(9):834-9.
Song L, Tuan RS.
MicroRNAs and cell differentiation in mammalian development.
Birth Defects Res C Embryo Today. 2006 Jun;78(2):140-9.
This is an interesting paper, which casts a new light on functional restoration of brain functions using neural stem cells. Unlike ES cells, which can theoretically give rise to any neurons in the brain, neural stem cells have a limited potential of differentiation repertories. In this paper, neural stem cell graft did not replace neurons but helped normalize the function and signals of host neurons with a genetic problem. This sort of supporting function has received some expectations for cell graft therapy for spinal cord injury, for which the injection of cells may somehow contribute to rejuvenation of the tissue environment and create more favorable conditions for reinnervation.
It is worth testing whether a similar strategy is applicable to human neurodegenerative diseases.
Smith et al. have shown the preclinical potential for antisense phosphorothioate oligonucleotides as therapeutic agents for neurodegenerative diseases, focusing on anti-SOD1 for ALS. Delivered as a saline solution via Alzet miniosmotic pump directly into the CNS, the antisense oligos are detectable throughout the CNS at micromolar (pharmacologically relevant) concentrations, most notably in the lumbar spinal cord region that is the earliest affected area in ALS rodent models. Similar results are obtained from infusion of an irrelevant tracking oligo directly into the brains of rhesus monkeys.
Dose-dependent reduction of SOD1 mRNA is shown from transfection studies using cultured cells, but similar dose-dependent effects data are not shown from in vivo pump delivery of antisense SOD1 into animals. That’s too bad: it’s not clear what determines an “optimum dose” for rat or monkey CNS, and whether reduction in SOD1 was the only readout for determining the optimum dose, and the authors don’t report whether any toxicity effects or tolerability issues were observed at any of the tested doses.
Using a SOD1 transgenic rat model as a test for therapeutic efficacy, the authors show that a month of pump-delivered antisense does nothing to delay onset or early weight loss of the treated animals—similar to saline. The authors do report a temporary shift in the survival curves (50 percent of treated rats are alive at a date when only 10 percent of control rats are still alive), but these curves converge at the end stage of disease, with steep losses in the treated group in the last 2 weeks, resulting in no clear extension of life. These data are consistent with the inability of the antisense to delay onset or weight loss stages of disease.
The story as a whole is interesting and potentially promising. The Alzet pump delivery methods, requiring surgery for implantations and changes, are problematic and could limit the usefulness of such studies. And, why is there an apparent limit to reducing SOD1 to 40 percent of baseline? Is this an absorption issue, or other limit of the technology, or does this say something else about the expression of SOD1 gene and its mRNA? Either way, it’ll be awhile before this type of experimental therapy is applicable to Alzheimer disease, in which the time course of disease development is more gradual and over greater periods of time (even in animal models), as compared to ALS.