Just one decade after its discovery, RNAi technology has not only developed in leaps and bounds but also found its way into human gene therapies and eyed for by biotech companies worldwide in the promise of far-reaching therapeutic applications. This paper is about the progress of research in RNAi in just a few years that led to many current and promising prospects for human therapies based on these cellular mechanisms. Along with its bulk of potential, RNAi-based biotechnology poses a list of questions on how to address problems mostly on aspects of triggered immune responses and safety from side effects when administered exogenously.
History
It was not after 1980 that RNA has been acknowledged as a molecule with an active role in cellular biology. The common notion that RNA was a passive carrier of genetic information from DNA to carry out protein synthesis has been dispelled after the discovery of catalytic RNA. Tom Cech and Sidney Altman both garnered the Nobel Prize for this study. In those days, genes were first defined via description of their mutant phenotype. This was called Forward Genetics, where the phenotype of the resulting mutant gives clues to the function of the gene. At the pace the sequencing of genomes are going now, thousands of genes have been identified yet we only know little of its function. It is therefore more practical, effective and less time-consuming to carry out genetic analysis that proceeds from genotype to phenotype called Reverse Genetics. This can be achieved by a number of gene-manipulation techniques including homologous recombination in embryonic stem cells and antisense approaches.
Ten years ago, Andrew Fire and Craig Mello discovered that (double stranded) dsRNA can trigger silencing of complementary (messenger) mRNA in Caenorhabditis elegans just after its genome sequence was completed in 1998. A new term, RNAi or RNA interference was born. The artificially generated (short interfering) siRNA, which were just short dsRNA, followed soon after that to demonstrate the same mechanism also occurs in mammalian cells, albeit not all the time. The discovery of RNAi related post-transcriptional gene silencing (PTGS) in plants to the activity of dsRNA. They found that the presence of just a few dsRNA molecules was enough to suppress the expression of a gene that was homologous to the dsRNA almost completely.
Many papers followed suit soon after this discovery; and a second wave of studies is currently blossoming as many developments in RNAi technology on mammalian systems are made. This time researchers are not only focused on elucidating gene functions but also developing genetic and antiviral therapeutics. The pace of discovery has quickened for RNAi technology and biotech firms are keen on taking advantage of breakthroughs for new applications for this emerging field.
What is RNAi?
RNAi is a specialized defense mechanism of RNA degradation that eliminates foreign RNA molecules, specifically those that can be identified by virtue of their appearance within the cell in double-stranded form. This can be found in many organisms including single-celled fungi, plants, plants, worms and mammals. The high occurrence in nature suggests a genetically conserved and evolutionarily ancient defense mechanism.
Research findings in plants determined that RNAi protects the plant cells against RNA viruses. In other organisms, it is thought to be a defense against the proliferation of transposable elements that replicate via RNA intermediates. There are a lot of kinds of plant viruses and transposable elements that produce double-stranded RNA, at least transiently, throughout their life. RNAi not only helps to keep such infestations in check, but also provides researchers with a powerful experimental technique to turn off or suppress the expression of individual cellular genes.
Most RNAi effector molecules consist of approximately 20 to 30 nucleotides, which forms a complex with the protein components of the RNA-induced Silencing Complex (RISC). Its catalytic core is called AGO2 in plants and animals, except for single-celled organisms. AGO2 is under the Argonaut family of proteins. There are two ways they do their jobs of gene silencing: First is Post-Transcriptional Gene Silencing (PTGS) under which there are two main mechanisms (Direct Sequence-Specific Cleavage, and Translational Repression & RNA degradation; and second is Translational Gene Silencing (TGS).
How is the RNAi mechanism activated? TGS has been demonstrated in fission yeast, a number of plants species, animals and most recently in human cells. In S. pombe a mediator called RNA-induced Transcriptional Silencing (RITS) complex exists. This contains AGO1, the chromodomain protein Chp1 and the Glycine and Tryptophan (GW)-repeat-containing protein Tas3. The specific mechanisms of TGS in mammalian cells and is still currently being debated, wherein AGO1 and AGO2 seemed to play an important role in the process of small-RNA-directed silencing.
Many of the endogenous small RNAs found in organisms originate from the transposons, viruses and repetitive sequences. These are characterized by their interactions with the PIWI subfamily of Argonaut proteins, and appropriately named piRNA or PIWI-interacting RNA. Recent studies have uncovered a new class of siRNA, endo-siRNA or esiRNA, which have been found in D. melanogaster gonads and somatic tissue and mouse oocytes. The proposed function of these endo-siRNAs is the regulation of retrotransposon movement in mice. Several families of small RNAs have been found to be expressed in fungi, plants and animals, but they haven’t yet been observed in mammals. Perhaps piRNAs act through different pathways, separate from siRNAs and miRNAs, and could one day offer alternative targeting strategies for therapeutics.
How is the RNAi mechanism being used?
Exogenous introduction of siRNA could target cellular genes via the PTGS mechanism. They could introduce the siRNAs into the cell by transfection, after which they enter the RISC complex directly, or they could otherwise be generated within the cells through gene expression by the use of vectors containing the promoters Pol2 or Pol3. These triggers are not present in S. pombe, but exist in plants and animals in the form of miRNAs or as (Short Hairpin) shRNAs. These are cleaved into smaller RNAs, around 20 to 25nt long, by the Drosha and/or Dicer enzymes. The passenger strand will be cleaved by AGO2 if the two RNA trigger strands are completely matched. This will then leave behind a single-stranded guide sequence that acts as the recognition template of the targeted gene sequence by RISC.
The mechanism above is taken advantage of by researchers who are finding therapies using gene silencing techniques. Most of the imminent applications in therapeutics propose using direct introduction of synthetic siRNAs. There are several advantages in using chemically synthesized molecules for this: chemical modifications can be introduced to increase stability, promote efficacy, block binding to unintended targets that contain sequence mismatches (specific off-target effects), and reduce of abrogate potential immunostimulatory effects (general off-targets effects). The said effects of the siRNAs are temporary, while the miRNAs and promoter-expressed shRNAs are good candidates for molecules that can mediate long-term silencing by administering it only once.
What are the Methods for Delivery?
There are two main strategies of delivering these chemically synthesized siRNAs. The first one is through non-viral means and the second is through delivery of shRNA-encoding genes by genetically engineered viruses that will, upon entry into the targeted cells, transcribe the siRNAs. Effective delivery into the targeted cells is crucial in the quest for developing RNAi-based therapeutic applications.
In one of the Non-Viral Delivery method the two polymers, atelocollagen and chitosan, have been examined for their properties that are useful for the delivery of siRNAs. Chitosans, for example, have mucoadhesive properties that make them a viable agent for intranasal delivery. There are studies that have been made on delivery of siRNAs to bronchiolar epithelial cells in mice and non-human primates, also in intravaginal delivery of lipid-encapsulated siRNAs (tests on HSV-2 treatment). In fact siRNA delivery to mucosal membranes seems to be a promising and effective approach.
Viral delivery approaches offers alternative means of triggering the RNAi mechanism. This is achieved mainly through the promoter-expressed siRNA sequences processed from shRNAs or miRNA mimics. Viral vectors are inserted with the genes encoding the hairpin structures usually controlled by Pol2 or Pol3 promoters. This is a very promising lead for therapies of chronic viral diseases like Hepatitis Viruses and HIV since vector delivery of a single administration could trigger long-term expression of the RNAi.
Designing a delivery system for a specific target requires stringent parameters such as tolerability, the length of time of expression, the ability to regulate expression and targeting and efficacy. There is always the risk of incurring mutations in the viral sequences that may trigger aberrant gene expression and cause insertional mutagenesis. The great advantage of having viral vectors is that it yields a prolonged expression of the therapeutic gene, hence only a small dose is enough, which is ideal for chronic anti-viral therapies. It can also transduce both dividing and non-dividing cells. It important to bear in mind that even though the engineered viruses are non-pathogenic, they are still potentially immunogenic and may induce adverse reactions from the subject. Just like any other therapeutic drug would be, any therapeutic gene when expressed in large quantities has the potential to cause toxicity and immunogenicity. All things considered, it is always good to remember that there is no ideal delivery system for every application; rather, the delivery method needs to be tailored to the application.
RNAis and Gene Silencing in Human Therapeutics.
RNA interference has proved to be particularly effective in C. elegans and D. melanogaster models, but in mammalian cells the same methods used in the previous models seem to elicit a nonspecific response until now. Articles in Nature reveal that RNAi can work in mammalian models. Research in the University of Gottingen by Thomas Tuschl and colleagues reveals that although introducing dsRNA into mammalian cells lead to a nonspecific response, introducing the siRNAs themselves into the cells can initiate RNAi.
There are a number of ongoing research and RNAi therapeutic clinical trials being done by pharmaceutical companies that wish to address genetic and viral diseases in humans. Of all the RNAi-based therapeutics and siRNA protocols tested in human clinical trials existing today, the first to be granted Investigational New Drug (IND) status is the Vascular Endothelial Growth Factor (VEGF)-targeted siRNA called Bevasiranib by Acuity Pharmaceuticals. The study aimed to treat Wet Age-related Macular Degeneration. Initial trials on mouse models showed reduced neovascularization that resulted from the downregulation of the gene Vegf after injection of the siRNA in the eyes. This is now in the Phase 3 clinical trials. Treatment of Diabetic Macular Oedema with the same delivery method is currently in Phase 2.
Treatment of AIDS lymphoma has already started phase 1 clinical trials in Duarte, California. The approach uses HIV-based lentiviral vectors inserted with the shRNA gene that targets Pol3 promoter-expressed shRNA targeting the HIV tat and rev shared exons. Also in the engineered vectors are other RNA-based anti-HIV genes. The delivery method is through autologous bone marrow transplantation to treat AIDS-related lymphomas.
There are many more studies underway hoping to develop therapies against viruses, and genetic diseases that are both acquired and inherent. Still, many more mechanisms that are involved in the different pathways that siRNAs use remain a mystery. Some effects, both positive and negative might not be brought about by the expected mechanism. For example, in the Wet Age-related Macular Degeneration study by Acuity Pharmaceuticals, other researchers demonstrated that the decrease in vascularization could probably not be a consequence of an siRNA-specific effect on angiogenesis, but rather a Toll-like Receptor 3 (TLR3) non-specific activation and its downstream counterparts that reduce the expression of VEGF.
The regulatory complexities of miRNAs should also be taken into consideration when either ablation or restoration of miRNA finction is being considered in a therapeutic setting. A single miRNA can regulate the levels of hundreds of proteins, raising autionary flags about the consequences of downregulating or ectopically expressing even a single miRNA species. Indeed, we haven’t yet fully mapped the pathways that all the miRNAs affect and disturbing the levels of expression in one would surely bring a cascade of other changes in the levels of other molecules that are involved.
RNAi in the Biotech Business World
A definite and workable RNA inhibition-based therapy on humans hasn’t yet been commercialized, but the list of Biotech and Pharmaceutical companies working on these are growing year by year. Many of them want to lead in the race for the first to develop the new-generation therapies, and intellectual property (IP) clashes between big companies like Merck/Sirna and Alnylam could be found in the news. It is hard to draw the line between what is patentable and not in RNAi therapy development and in the meantime, the search for more applications of these drugs will continue. Even the Chinese Biotech company, Shanghai Genomics, is offering its services in RNAi therapeutics research. There is high market value in the promise that gene silencing approaches offers, and with so many players around to compete there might be a breakthrough closely at hand.
Problems Ahead
There are still the problems primarily concerning safety. There are records of death in mice after Pol3 promoter-driven expression of shRNAs in the liver were administered, and the mechanism as to how this happened is still under scrutiny. As mentioned above, indications that other factors in the RNA inhibition process can be saturated by over expression of exogenous siRNAs, which can then recruit them from their cognate cellular miRNAs. The problem lies, again with the limited knowledge in the miRNA pathways and the consequences of its disturbance.
A way to address the problem and at the same time detect what is going on in the cell is to administer the lowest possible dose. Then with the help of microarray technology, monitor the expression of non-targeted genes as well as targeted ones. This way, they could get an idea of how off-target the range of the effect of the silencing could be.
There is also the problem on the ability of siRNAs to stimulate immune responses based not only on specific sequences but also on structure, the type of delivery system and the cell type. These factors are all essential for the RNAi to work and changing one because of findings of TLR stimulation and immunogenesis might not get the same effect as the originally planned design.
Future Work
Despite the current setbacks that the technology is facing, the potential candidates for RNAi therapy targets are so extensive that one could not simply ignore them. Genetic and viral diseases that are hard to cure has been given a new and fresh perspective on how to tackle the problems surrounding these. HIV, tumor suppression and cancers, geriatric diseases, respiratory diseases and many more have a chance to be addressed in the future. The technology is still young but the progress in research in this area has been rather quick since its discovery only a decade ago.
RNA inhibition is not an accepted form of therapy. Yet. In the coming years however, more siRNA pathways and mechanisms will be uncovered and little by little the mysteries surrounding gene silencing will be elucidated. For now, the race from the lab to the clinic is on for the many hopeful Biotech and Pharmaceutical players.
