Technology Insight: Therapeutic RNA Interference - How Far from the Neurology Clinic?

Pedro Gonzalez-Alegre; Henry L Paulson*


Nat Clin Pract Neurol. 2007;3(7):394-404. 

In This Article

Challenges Ahead

The most important information gained from preclinical studies has been the demonstration that an RNAi approach, using various delivery strategies, is feasible for a wide variety of neurodegenerative diseases ( Table 1 ). Studies have also, however, highlighted some of the challenges that the RNAi field faces before it is in a position to conduct human trials. These challenges include achieving efficient delivery, reducing possible off-target effects and adverse consequences of therapeutic RNAi, and developing reliable measures of efficacy in human studies.

It is no coincidence that all but one of the in vivo preclinical studies mentioned above were performed with intracellularly expressed RNAi delivered via recombinant virus. Although synthetic, chemically modified siRNAs have worked well in systemic delivery to other organs, the blood-brain barrier makes this kind of delivery a more daunting task in brain disorders. Direct intraparenchymal injection of neurotropic virus circumvents the blood-brain-barrier issue.[61] In addition, virally delivered shRNAs provide continued intracellular expression of the RNAi effector molecule, something that will probably be required in chronic neurodegenerative disease states. Although viral delivery of therapeutic RNAi has been highly effective in rodent models, the reader should keep in mind that the mouse striatum or cerebellum can be transduced with one or a few small-volume injections. The human brain, being orders of magnitude larger, will require a more complex administration approach. Additional studies in the nonhuman primate brain, or possibly porcine brain, should help bridge the gap between preclinical rodent studies and human trials. Furthermore, improvements in the large-scale production of clinical-grade viral vectors are likely to be required if this technology is to successfully enter the clinical arena.

Although viral delivery of RNAi for specific neurodegenerative diseases is likely to be developed first, continuing advances in modifications of synthesized siRNAs could eventually lead to successful CNS delivery.[28,62,63] Chemical modifications at specific points along the siRNA ribose backbone greatly stabilize siRNAs without unacceptably reducing potency. Likewise, conjugation to cholesterol or incorporation into liposomes stabilizes siRNAs and facilitates delivery to tissues. Tethering the siRNA to antibody fragments or to RNA aptamer motifs that bind specific cell-surface receptors can both enhance and restrict delivery to specific cell populations in the CNS. In addition, encasing siRNAs in customized nanoparticles that are coated with specific ligands can promote delivery to specific cell populations. The distinct advantage of synthetic siRNAs for many applications is their transient nature; any unexpected adverse consequences can be countered by halting further administration. The same cannot be said of RNAi-expressing virus, which would be expected to continue expressing shRNA against the target gene. For this reason, efforts are underway to adapt existing regulable expression systems for RNAi viral delivery so that expression in humans might be titrated up or down by varying the administration of a simple compound.

To date, preclinical studies in animal models have focused on efficacy, with less attention having been paid to the potential adverse consequences of exogenous manipulation of the RNAi pathway. In a recent report, virus-mediated RNAi delivered to hepatocytes in vivo caused significant toxicity by impairing aspects of the endogenous miRNA pathway.[64] Similar effects could occur in neurons—postmitotic cells in which endogenous miRNAs play a major role.[25,65] Broadly speaking, adverse consequences of RNAi can derive from the abnormal presence of a dsRNA in the target cell, the utilization of the endogenous RNAi pathway by exogenous dsRNA, and off-target effects caused by the specific sequences employed in the therapeutic construct ( Table 2 ).[30]

One possible adverse consequence of RNAi is the induction of an immunostimulatory response. This possibility is greater with exogenously delivered siRNAs than with intracellularly expressed shRNAs. The interferon response is a naturally occurring cellular defense mechanism against the entry of exogenous nucleic acids.[66] Double-stranded RNAs longer than ~30 bases are recognized by this protective pathway, triggering a molecular cascade that ends in global translational repression. Although dsRNA shorter than 30 bases was not expected to trigger this response, initial studies in various cellular systems yielded conflicting results,[67,68,69,70,71,72] suggesting the possibility of cell-type specificity. Studies in cultured mammalian neurons transduced with virally encoded shRNAs did not detect activation of this protective response.[39] Furthermore, assessment of the interferon response in mice that were overexpressing an shRNA that targets mutant SOD1 failed to show activation.[52] Moreover, even if triggered, the interferon response is usually transient and might not have notable long-term effects if an RNAi-mediating construct was to be administered only once. Repeated administration of synthetic siRNAs, however, could lead to periodic activation of this protective response. In future RNAi studies in the CNS, a systematic analysis of possible immunostimulatory responses, including but not limited to the interferon response, will be important to clarify the extent to which immune responses will act as a barrier to effective therapy.

A second class of potential adverse effects are those derived from saturation of the RNAi machinery by exogenously administered dsRNAs, which could interfere with the normal function of endogenous miRNAs that are critical for neuronal function. The need to carefully assess this possibility was underscored by a recent study that reported shRNA-induced abnormalities in dendritic arborization of cultured rat neurons.[65] Minor alterations in the miRNA pathway could lead to functional defects such as abnormal neuronal polarization or altered synaptic function. Although these defects might not lead to overt behavioral abnormalities in rodents, they could have profound effects in the more complex human brain. Development of appropriate measures of endogenous RNAi function that could be employed in the brains of different mammalian species would help to ensure that scientists advance with an acceptable level of safety toward human trials.

A third area of concern is potential off-target effects resulting from the specific sequence contained in the RNAi reagent. In choosing a target sequence and designing an RNAi molecule, informatics-based searches are routinely performed to exclude identical or highly similar sequences in other genes, which might inadvertently be affected. Even in the presence of significant sequence mismatches (particularly in the 3' half of the antisense strand), however, other unintended mRNAs could be suppressed.[68,73,74] The RNAi field still lacks a completely reliable algorithm to predict unintended effects. Before applying a specific dsRNA to the human brain, researchers should consider performing experiments in cultured human neural cells with gene microarray analysis to identify the pattern of neuron-expressed genes that are unexpectedly suppressed by the specific sequence used.

It can be assumed that most RNAi reagents will have identifiable off-target effects. From the perspective of therapeutic RNAi, however, what really matters is whether these off-target effects cause in vivo toxicity or otherwise impede the beneficial effect of the RNAi reagent in suppressing the target disease gene. To lessen the risk of these potential adverse consequences, we must strive to use the lowest clinically effective doses of RNAi reagents that have been engineered to be processed as efficiently as possible and that also favor incorporation of the guide strand into RISC. This goal can be accomplished now that the rules governing strand selection are better understood,[75,76] the engineering of shRNAs to mimic endogenous miRNA-like structures has improved,[77] and the capacity to control expression with tissue-specific and regulable promoters is at hand.[78]


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