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A New Gene Silencing Technique Enters the Pipeline
An Overview of Gene Silencing Progress
Researchers associated with Isis Pharmaceuticals have published two articles on the effectiveness of a new technology designed to selectively silence the Huntington’s disease gene. Researchers were able to chemically modify single-strand small interfering RNA to achieve the efficiency of double-stranded short interfering RNA while maintaining the simplicity of the antisense oligonucleotide approach.
Two methods of gene silencing have been moving through the pipeline of potential HD treatments, antisense oligonucleotides and RNA interference.
Antisense oligonucleotides (ASOs) are synthetic nucleic acids whose base sequences are complementary to the target gene's messenger RNA. They are paired with and bind the messenger RNA, degrading it through an enzyme called RNase-H. so that the instructions to make the huntingtin protein are not translated and the protein is not made.
ASOs involve single strands of RNA so they can penetrate the cell membrane. For this reason, ASOs can be made into drugs. There is still a problem of delivering them to the brain to treat Huntington’s or other neurodegenerative diseases caused by a dominant gene. The current approach is to have them pumped into the brain through the cerebrospinal fluid.
In June, we published a report on research by Dr. Donald Cleveland and his team which showed that a ‘Huntington’s holiday’ approach was an effective treatment for several mouse models of HD. In this approach, ASOs were administered intermittently. The technique used was non-allele specific, meaning that both the normal huntingtin’s protein gene as well as the HD gene were silenced. The idea is to silence the disease gene long enough to allow the brain to catch up on clearing away the toxic protein but not so long that the brain is negatively affected by the reduction of the normal protein. Dr. Cleveland commented that this approach could go to clinical trials within eighteen months. (Go to http://www.hdsa.org/research/aso.html to read the article.)
It is also possible to use ASOs to selectively silence the HD gene. Dr. David R. Corey and colleagues have been able to develop ASO formulations that bind to the extra CAG repeats, making use of the abnormal structure of the expanded section of repeats. Their formulations inhibited the HD allele but did not affect the normal huntingtin protein allele. Other genes which normally contain stretches of CAG repeats were also unaffected. The researchers wanted to improve the level of sensitivity they achieved however, (between 4 and 8 fold) and that is what prompted their research with single stranded RNA interference.
Dr. Michael Hayden and colleagues made use of single nucleotide polymorphisms (SNPs) found on the HD alleles but not on normal alleles to selectively silence the HD gene while allowing the normal allele to express itself. This technique was first used in HD RNAi research by Neal Aronin and Robert Friedlander (see below). By using this technique in ASO formulations, two limitations can be overcome. Fist, the results are more highly selective. Second, because intervention takes place at an earlier part of the process of gene expression, SNPS in the intron can be used, not just the ones in the exon as is RNA interference. Introns are sections of DNA that are not used in coding for a protein and they are removed during gene transcription. With greater possibilities of finding SNPs, it seems likely that all or virtually all HD patients could benefit from this technique. However, as with double stranded RNAI, this is personalized medicine, requiring DNA analysis of individual patients and clinical testing of each formula.
The HD community has been encouraged by Isis’s Phase I clinical trial with ALS patients who have a dominant gene which has caused their disease. Most ALS is not caused by this one gene but a small percentage of patients do fall into this category so there is hope that a gene silencing treatment would work for them. Isis presented the results of the Phase 1 study at the American Academy of Neurology meeting in April 2012. This was Isis’s first experience with administration of antisense drugs in humans through an injection into the spinal fluid. The drug was well tolerated and no safety issues were found. This successful trial is paving the way for an antisense trial for Huntington’s disease (according to conversations with an Isis representative).
Double-stranded short interfering RNA
RNA interference can also produce gene silencing. The potential RNA interference treatments that are moving through the pipeline for Huntington’s disease involve double-stranded RNA that silences the target gene by prompting the cell’s own defense mechanisms against foreign RNA such as the RNA in viruses. Double-stranded RNA molecules that match the RNA of the target gene are introduced into the cell. They are then cut into small fragments by an enzyme called Dicer. The double strands separate; the passenger strand is degraded by the RNA-induced silencing complex (RISC) which also seeks out the cell’s own complementary messenger RNA. Within the complex is the Argonaut2 protein which degrades both the guide strand and the complementary messenger RNA, thus silencing the message to produce the gene.
Dr. Beverly Davidson showed the proof of principle for this technique with her studies in an HD mouse model. Drs. Neil Aronin and Robert Friedlander have developed ways of making the technique allele-specific through the use of single nucleotide polymorphisms (SNPs), small mutations that are commonly found on one allele but not the other. Since these SNPs differ from person to person, this would involve personalized medicine and each formulation of double-stranded short interfering RNA would have to be tested through clinical trials. Although it is estimated that most people with the HD gene do have a polymorphism on the HD gene which would allow allele specific silencing, some do not.
The small interfering RNA in this approach is double-stranded, making delivery more complicated. Not only does it have to get into the brain, there needs to be a method of getting it into the cells. There are two possibilities; one is to use a viral vector, a well-established technique where the short interfering RNA is inserted into viruses which are stripped of their harmful DNA. When the virus enters the cell, it carries the siRNA with it.
The other possibility is an approach being developed by Dr. Jan Nolta where the siRNA is delivered through mesenchymal stem cells. The idea here is that these stem cells will travel throughout the brain to attempt to repair damaged areas and cells and deliver the siRNA to the cells while doing so.
There is widespread interest in RNA interference as a treatment for a variety of diseases. RNAi treatments are in Phase I or Phase II for illnesses such as cancers, diseases of the eye, viral disorders, and kidney disease. Pharmaceutical companies Lundbeck and Alnylam are both working on developing siRNA as a potential treatment for Huntington’s disease.
Single-strand small interfering RNA
The new research shows that single stranded short interfering RNA (ss-siRNA) can be chemically modified to effectively silence the HD gene while allowing the normal huntingtin’s gene to express itself. Ss-siRNA had not been previously considered for gene silencing therapy because, although it can be used for gene silencing in cells, it doesn’t perform as well as double-strand siRNA and it is inert in animals because of a lack of stability.
In the new studies, researchers from Isis Pharmaceuticals, the University of Texas Southwestern Medical Center, and the University of California at San Diego describe how they were able to stabilize ss-siRNA by adding a phosphate to the 5 prime terminus (end) of the strand.
In the first study (Lima et al), the researchers used an entirely complementary strand of ss-siRNA, achieving the silencing of various target genes in cells and in the peripheral tissue of mice. The silencing functioned through a siRNA-like pathway involving cleavage by the argonaute 2 protein (see the last column of Figure A). The ss-siRNA was packaged in saline solution. Complex lipid formulations were not needed for effectiveness as they are with double-strand siRNA, thus reducing the likelihood of toxicity. Since single strands of short interfering RNA were used, as with ASOS, there was no need for a viral or other vector as is the case with double stranded siRNA. Further, the use of single-strand siRNA, reduces the likelihood of off target effects as compared to double-strand siRNA.
In the second study (Yu et al), the researchers targeted the HD gene in cells and animals, using a similar formulation with one major difference. Instead of using a strand which was entirely complementary to the messenger RNA of the target gene, they introduced a mismatch in one of the base pairs. They were able to silence the HD gene while allowing the normal huntingtin’s protein gene to express itself in both cells and in a knockin mouse model of HD. The mice had the ss-siRNA continuously infused into the brain through the cerebral spinal fluid of the right lateral ventricle for four weeks. Importantly, the HD protein was reduced throughout the entire brain. Of course, the mouse brain is very much smaller than the human brain, but the widespread distribution is still encouraging.
Figures A and B are from the Yu article and used by permission from Dr. David Corey and colleagues
As in the first study, gene silencing took place using RNAi machinery. However, the pathway through which this was achieved was different than in the first study. Argonaut 2 (AGO2) could not cleave the messenger RNA; there was no reduction in messenger RNA or the normal protein but there was a potent reduction in the HD protein -- so how did this occur? According to the researchers, gene silencing takes place through a micro RNA like pathway. Multiple single strands of siRNA bind in complex with AGO2 to the CAG repeat section of the target messenger RNA. Instead of degrading messenger RNA and blocking transcription, the translation of the protein is blocked (see Figure B).
How was allele-specific silencing achieved? The researchers suggest that it is through cooperative inhibition. The longer the CAG repeat stretch, the more sites there are to which the ss-siRNA can bind. For example, messenger RNA from an HD gene with 69 CAG repeats can bind 0 or 10 single siRNA strands but mRNA from a normal allele with 17 repeats could bind no more than two. Using the technique in a variety of cells with a normal and an HD gene, the researchers were able to achieve selectivity with as few as 29 CAG repeats difference in the two alleles, 44 on the HD allele and 15 on the normal allele.
The mismatch approach resulted in a 30 fold selectivity compared to the 4-8 fold selectivity the same researchers had achieved through ASO silencing. The selectivity was comparable to that achieved by other researchers using double-strand DNA and SNPs to differentiate between the two alleles.
Both sets of researchers call for further development and improvement in their ss-siRNA formulations. We can also expect to see further testing in animals.
Comments on the advantages of the approach
"Double-stranded siRNA drugs require complex formulations to achieve sufficient delivery for systemic activity. This requirement has severely limited the development of safe and effective drugs that work through the RNAi pathway. In contrast, single-stranded RNA-like antisense drugs can be administered subcutaneously and distribute to tissues without the need for formulations. In addition, approaching RNAi with single-stranded drugs eliminates the need for the complementary strand, the 'sense' strand, which limits the risk of adverse effects, and reduces the cost of manufacturing," said Stanley T. Crooke, M.D., Ph.D., Chairman of the Board and Chief Executive Officer of Isis. "At Isis, we have made significant breakthroughs in oligonucleotide chemistry developing single-stranded RNA-like antisense drugs that can work through many different antisense mechanisms, including RNaseH, splicing, and now RNAi."
Researcher David Corey, Ph.D., Professor of Pharmacology at University of Texas, Southwestern Medical Center commented on both articles. "Together, these data provide compelling evidence that single-stranded oligonucleotides can be designed to exploit the RNAi pathway and silence gene expression of specific mRNAs in target tissues. In our initial experiments with animals, the compounds were well-tolerated even when used at high doses. Single-stranded antisense technology provides a new strategy for harnessing the RNAi pathway. It combines the potential of RNAi with the favorable drug properties of single-stranded nucleic acids."
"Working with our colleagues at the University of Texas Southwestern and UCSD, we showed that, using ss-siRNAs, we could selectively target and specifically silence the gene responsible for the disease-causing form of huntingtin protein and exploit the RNAi pathway to produce microRNA-like effects," continued Dr. Crooke. "We believe that this work lays the foundation for the development of a robust, drug discovery platform that takes advantage of using single-stranded RNA-like antisense drugs that harness the power of the RNAi pathway for gene silencing."
Drs. Beverly Davidson and Alex Monteys discuss the implications of both articles in an introductory essay and conclude that “These studies introduce ss-siRNAs as an exciting new tool in our arsenal to silence disease-causing genes for therapeutic applications and basic research.” They also note that, “It is also possible, given their stability and ability to enter cells without lipid formulation, that they may find utility as a sponge for disease-related miRNAs. Given the noted tolerance of the liver to high doses of the ss-siRNAs, they could conceivably be used to inhibit miRNAs involved in disease pathogenesis.” Since this new technique holds promised not just for diseases caused by dominant genes but diseases caused by micro RNA (such as some cancers and cardiovascular diseases), there is a clear economic incentive for further development of this potential treatment.
Isis is now evaluating competing approaches to treating Huntington’s disease through gene silencing but hopes to identify a development candidate next year on which to begin conducting toxicity studies to enable an Investigational New Drug (IND) application to be submitted to the FDA (personal communication).
It is too soon to predict which approach from which pharmaceutical company will result in the first gene silencing trial. It is even possible that more than one approach will be brought to trial. Assuming that preclinical studies contain to show efficacy and safety, we are likely to see a trial of at least one approach within the next two years or so.
Jeffrey B. Carroll, Simon C. Warby, Amber L Southwell, Crystal N Doty, Sarah Greenlee, Niels Skotte, Gene Hung, C Frank Bennett, Susan M Freier and Michael Hayden. “Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene: allele-specific silencing of mutant huntingtin.” Molecular Therapy 2011 Dec;19(12):2178-85.
Beverly L. Davidson, and Alex Mas Monteys. “Singles Engage the RNA Interference Pathway.” Cell 2012 August;150(5): 873-5.
Keith T. Gagnon, Hannah M. Pendergraff, Glen F. Deleavey, Eric E.Swayze, Pierre Potier, John Randolph, Eric. B. Roesch, Jiyoti Chattopadhyaya, Masad J. Damha, C. Frank Bennett, Christophe Montaillier, Marc Lemaitre, and David R. Corey. “Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat.” Biochemistry 2010 November 30;(47):10166-78.
Keith T. Gagnon, John Watts, Hannah M. Pendergraff, Christophe Montaillier, Danielle Thai, Pierre Potier, and David R. Corey. “Antisense and Antigene Inhibition of Gene Expression by Cell-Permeable Oligonucleotide–Oligospermine Conjugates.” Journal of the American Chemical Society 2011, 133 (22), pp. 8404–8407.
*Walt F. Lima, Thazha P. Prakash, Heather M. Murray, Garth A. Kinberger, Wenvu Li, Alfred E. Chappell, Cheryl S. Li, Susan F. Murray, Hans Gaus, Punit P. Seth, Eric E. Swayze, and Stanley T. Crooke. “Single-Stranded siRNAs Activate RNAi in Animals.” Cell 2012 August;150(5): 883-894.
*Dongbo Yu, Hannah Pendergraff, Jing Liu, Holly B. Kordasiewicz, Don W. Cleveland, Eric E. Swayze, Walt F. Lima, Stanley T. Crooke, Thazha P. Prakash, and David R. Corey. “Single-Stranded RNAs Use RNAi to Potently and Allele-Selectively Inhibit Mutant Huntingtin Expression.” Cell 2012 August;150(5):895-908.
- Marsha L. Miller, Ph.D., October 10, 2012