A hit and a miss for RNAi
For many neurodegenerative diseases where the accumulation of misfolded, toxic proteins causes cell death, using RNA interference and antisense approaches to knock down the expression of specific proteins is a promising approach. That is exactly the path taken by Don Cleveland and colleagues at the University of California at San Diego, who report that delivery of synthetic antisense oligonucleotides directly into the CSF reduces levels of disease-causing SOD1 mutant mRNA and protein in the brain and spinal cord in a rat model of ALS. While the treatment did not delay onset of motor neuron disease, it did slow progression. The studies are a prelude to clinical trials in humans, which the researchers hope to start within a year, according to a press release on the study. The work, which was published online July 27 in the Journal of Clinical Investigation, offers a therapeutic strategy to downregulate almost any CNS protein.
One barrier to successful use of antisense oligos in the CNS is the problem of delivery. The researchers, led by joint first authors Richard Smith and Timothy Miller, tried delivering the oligos by osmotic pump into the cerebral ventricles. This method is already used in humans, they note, to deliver pain medication. Studies suggest that from the cerebral ventricles, the oligos would circulate in the cerebrospinal fluid, which bathes all regions of the CNS. Indeed, the authors showed that after a 14-day infusion in rats or rhesus monkeys, micromolar concentrations of oligonucleotides turned up in the brain parenchyma, as well as both the upper and lower regions of the spinal cord. They found oligonucleotides in both lumbar motor neurons and in non-neuronal cells.
Smith and colleagues then tested the ability of antisense SOD1 oligos to reduce protein levels in transgenic rats expressing the human G93A SOD1 mutant. These transgenic animals are a widely used ALS model. Even though the animals express very high levels of mutant SOD1 (5-10 times higher than endogenous SOD1), they effected a 40-60 percent reduction in mRNA and an approximately 25 percent reduction in protein when they administered a human-specific oligonucleotide. When they started infusion treatment in 65-day-old rats (~30 days before disease onset), they observed no change in onset, but the treatment did seem to slow progression, delaying the emergence of severe symptoms from day 122 to day 134, a 37 percent extension compared to the normal course of the disease.
Previous studies of viral delivery of antisense SOD1 in animals also showed promising results (Raoul et al., 2005; Ralph et al., 2005; Miller et al., 2005), but intraventricular delivery of oligonucleotides presents some advantages. First, the treatment can be easily regulated, with doses readily increased, reduced, or stopped. Also, in contrast to viral delivery schemes targeted only to neurons, oligonucleotides in the CNS get into other cells as well. This feature may be particularly important for ALS, where the expression of SOD1 in both neurons and surrounding glia is important in disease onset and progression (see ARF related news story and Boillee et al., 2006)
What of the application to other diseases? When the researchers infused antisense oligonucleotides to two Alzheimer disease targets, presenilin-1 and GSK3β, they saw a reduction in mRNA for the two proteins in the right frontal/temporal cortex after 14 days. While this does not ensure that protein levels would be reduced, the results suggest that the widespread delivery of oligos throughout the CNS could open opportunities to treat a number of diseases.
Animal toxicity studies are now under way in preparation for a planned phase I of the SOD antisense oligo in humans. If the treatment proves safe, it could open up opportunities to knock down many more targets. The authors cite, for example, proteins such as the amyloid-β precursor protein, β-secretase, tau, and presenilin-1 for AD, or huntingtin for Huntington disease, as targets of interest for future studies.
Any future trials will have to consider the specter of side effects. For RNA interference therapies, the possibility of off-target effects raises safety concerns. In particular, a recent report showed that the administration of viral vectors that produce short hairpin RNAs (shRNAs, the precursors to small interfering RNAs) can be fatal to mice (Grimm et al., 2006): By overwhelming the cellular machinery that produces endogenous microRNAs, the shRNAs perturb gene expression generally, which leads to cell death.
Now, another paper, this one from Bernardo Sabatini and colleagues at Harvard Medical School, shows an additional off-target effect of shRNAs that is specific to neurons. In work published in the Journal of Neuroscience on July 26, Sabatini, Veronica Alvarez, and Dennis Ridenour show that expression of shRNAs in neurons interferes with dendritic spine structure and function, and can result in decreases in synapse number. (For a complete description of off-target effects of interfering RNAs, see the comment below from Zuoshang Xu.)
The effects are independent of which mRNA is targeted, and even of the generation of siRNAs, but do depend on the sequence of the shRNA. In particular, shRNAs that induced an antiviral-type response, as measured by activation of an interferon target gene, affected neuron morphology and function, while shRNAs to the same protein that did not induce the interferon gene, did not. For research, the results demonstrate the need to use caution in using RNA knockdown data to probe protein roles in synaptic function and remodeling. More careful sequence selection and use of stringent controls must be the norm—the use of scrambled sequences is not sufficient, the authors say, and they stress the need for protein rescue experiments to ultimately prove the specificity of any observed effects.
For therapeutic RNAs, the same cautions will apply. However, synthetic antisense DNA oligonucleotides may have an advantage over shRNAs because, as Cleveland and colleagues argue, they do not require further processing and do not trigger an antiviral response.
Finally, what about gene therapy—a permanent fix for those genetic errors that give rise to many neurodegenerative diseases? For Alzheimer, Parkinson, and Huntington diseases, and others whose familial forms spring from dominant gain-of-function mutations, the strategy of adding a good copy of the gene will not work. Instead, the goal of gene therapy must be to actually repair the mutations in the gene in situ. While novel methods have been devised for therapeutic recombination in cells (see ARF related news story), the ultimate goal is to find techniques that work in vivo.
A new approach that uses adeno-associated viral vectors comes closest yet to this elusive prize. In a report published online in Nature Biotechnology, researchers from David Russell’s lab at the University of Washington in Seattle used the vector to repair a model mutation in a β-gal reporter gene in mouse liver in vivo. The group, led by first author Daniel Miller, also repaired a disease-causing mutation in the GusB gene encoding the liver enzyme β-glucuronidase, although the frequency of repair (one to two cells per 10,000) was too low to demonstrate a therapeutic effect. For that enzyme, they estimate a 10-fold increase in the efficiency of recombination would achieve a clinical effect. Though gene replacement in the CNS will be many times harder than correcting liver enzymes, the work provides a tantalizing promise of what may be possible, one day.—Pat McCaffrey.
Li J, Imitola J, Snyder EY, Sidman RL. Neural stem cells rescue nervous Purkinje neurons by restoring molecular homeostasis of tissue plasminogen activator and downstream targets. J Neurosci. 26 July 2006;26:7839-7848. Abstract
Smith RA, Miller TM, Yamanaka K, Monia BP, Condon TP, Hung G, Lobsiger CS, Ward CM, McAlonis-Downes, M, Wei H, Wancewicz EV, Bennett CF, Cleveland DC. Antisense oligonucleotide therapy for neurodegenerative disease. Journal of Clinical Investigation. July 27 2006; online publication. Abstract
Alvarez VA, Ridenour DA, Sabatini BL. Retraction of synapses and dendritic spines induced by off-target effects of RNA interference. J. Neurosci. July 26 2006;26:7820-7825. Abstract
Miller DG, Wang P, Petek LM, Hirata RK, Sands MS, Russell DW. Gene targeting in vivo by adeno-associated virus vectors. Nature Medicine. July 30 2006; advance online publication. Abstract