As more of the human genome is identified and characterized, new frontiers open for therapeutics. “The RNA interference (RNAi) research space continues to explode,” explains Susan Magdaleno, Ph.D., senior manager of scientists, RNAi technologies R&D, Life Technologies. “We are actually expanding into new content. Part of it is a result of the exploding data coming from next-gen sequencing—when you look at that content, it’s not messenger RNA. There are a lot of RNAs there that have yet to be characterized.”
“RNAi is a fast moving field—even incremental advances can result in high-profile publications,” agrees Paloma Giangrande, Ph.D., assistant professor, department of internal medicine, University of Iowa. “The promise of RNAi gets people excited, and any progress gets RNAi closer to the clinic.”
At CHI’s “RNA Interference: From Tools to Therapies” conference to be held in San Francisco next month, some of the latest tools and best practices captured from labs across academia and pharma will be described.
A noncoding RNA (ncRNA) is a functional RNA molecule that is not translated into a protein. ncRNA genes include highly abundant and functionally important RNAs such as transfer RNA (tRNA) and ribosomal RNA (rRNA). The number of ncRNAs encoded within the human genome is unknown, but Dr. Magdaleno says that her group’s efforts are to bring some functional analysis to ncRNA. “We’re starting to link ncRNA to disease, and it’s starting to grab the attention of investigators.”
ncRNA opens up novel targets for therapeutics. “Not all disease can be explained by changes in proteins, and there is only so much you can discover in proteins,” she adds. “Epigenetics is another area that is being greatly explored. ncRNAs might be influencing regulation at the epigenetic levels. The trick is to develop the tools that will allow robust functional analysis.”
Life Technologies developed a suite of integrated tools and workflows to discover, validate, and knock-down ncRNA to accelerate understanding of the function of ncRNA in the cell. At the conference, Dr. Magdaleno will describe requirements for using siRNAs to knock-down ncRNA and will highlight the application of siRNAs to investigate ncRNA function in regulating mitosis and apoptosis in normal and cancer cells.
“ncRNAs are not as well-characterized as messenger RNAs and many are not as abundantly expressed as messenger RNAs—low abundance is a challenge; many are only present under specific conditions. We don’t know why they are there, what happens to them, and whether we can develop therapeutic solutions based on ncRNA.”
Dr. Magdaleno’s group develops best practices for studying ncRNAs, which is not without challenges. “The ones we are focusing on are greater than 180 nucleotides. These are not highly characterized, there is no single fundamental source database of ncRNAs, nor is there an agreed upon definition of an ncRNA database. We are early in this field, but it’s important to determine functionality of these RNA species. We have the technology, but workflows still need to be figured out.”
Another quickly evolving area is Transkingdom RNA interference (tkRNAi), which takes a Trojan horse approach to solve one of the key challenges to RNAi therapeutics—delivery of the RNAi into the cell. Cequent Pharmaceuticals uses live attenuated bacteria to produce and deliver RNAi from the luminal side to the GI mucosa, allowing oral administration.
Johannes Fruehauf, M.D., vp of medical affairs, says that clinical trials are about to begin for the prevention of colon polyposis and for the treatment of inflammatory bowel disease. “tkRNAi technology deactivates specific disease-causing genes safely, and effectively uses nonpathogenic bacteria as an engine to produce and deliver RNAi directly into cells.”
Results were recently reported from large screening efforts characterizing the effects of tkRNAi on cytokine profiles and innate immunity. “We have an IND and will be starting a Phase I trial soon. It also has a biomarker aspect to it to allow for efficacy readout, but primarily we have to see that the treatment is safe.”
Polyposis is an orphan disease, Dr. Fruehauf says, and its population has been heavily surveyed. “We know that it’s genetic and that 100% of patients will develop cancer. What happens is that, starting in the teens, hundreds to thousands of polyps develop in the colon. Eventually, the whole colon must be removed. At present, there is no drug available to block polyps and make them regress.”
Dr. Fruehauf’s group targeted beta catenin, a well-known oncogene. “We have shown through several studies that if you block this gene, you can stop or block polyp formation. In our upcoming study, 18 people with this disease will be treated for one month. We’ll start with baseline biopsies and also collect end-point biopsies to see if there are any gene-expression changes in the gut.
“My talk will be on one specific aspect, immunogenicity, always an important question with RNAi drugs. We have done studies in mice to see if the vector itself suppresses gene-expression levels. We found that the effect of the carrier system is minimal, and the gene silencing effect is what is wanted. Oral delivery is the only way to get to the gut.”
A major hurdle for the clinical translation of siRNAs into effective therapies is delivery. “Many delivery approaches for RNAi center on nontargeted delivery—not delivering treatment to where it needs to go,” Dr. Giangrande explains. “The majority of these treatments end up in the liver, which is fine if you are targeting liver disease. Not so much if you need to target elsewhere.”
At the meeting, she will describe an RNA aptamer-based approach for the targeted delivery of siRNAs to prostate cancer cells. The aptamer-siRNA reagent, or chimera, is effective when administered systemically, she says, and is suitable for efficient chemical synthesis. Studies have shown that when administered systemically to mice bearing PSMA-positive tumors, the RNA chimera triggered tumor regression without affecting normal tissues.
“We are also developing approaches that can help us identify aptamers or RNA chimeras that would allow us to deliver siRNAs to many different cell types. At the meeting, I will summarize some of the work already published on prostate cancer. I will also present some recent progress on the development of approaches that may enable us to identify reagents that we can target to any cell.”
Dr. Giangrande’s group covalently links siRNAs to single-stranded, highly structured RNA oligonucleotides, known as aptamers. The resulting aptamer-siRNA reagent is allowed to bind to its target cancer-specific marker (receptors) on cancer cells, internalize, and deliver the therapeutic siRNA cargo to the RNAi machinery, resulting in silencing of the siRNA target gene.
“Reagents should be in circulation for longer periods of time for cancer. Increasing and optimizing pharmacokinetics (PK) without losing efficacy is another big challenge. We are developing ways to optimize the PK of these reagents. Our hope is to get these targeted cancer therapies into the clinic.”