microRNAs: new technologies, new drugs
RNA interference and pharma, or Along the Milky Way: from nucleotide to drugValery Yudin, "Weekly PHARMACY"
The discovery of RNA interference (RNA i) has significantly changed our understanding of biological processes and contributed to the creation of a new and powerful tool for biological research.
Technologies based on RNA interference are becoming an increasingly important platform for conducting research and studying medicines with significant potential, which allows us to significantly expand the points of their application.
Not so long ago, many well-known pharmaceutical manufacturers acquired or concluded cooperation agreements with small biotech companies in order to gain access to their technologies based on RNA interference in order to subsequently introduce medicines obtained using these technologies to the pharmaceutical market. Just like monoclonal antibodies before, drugs created on the basis of RNA I represent new ways of therapeutic effects, now proclaiming the beginning of a new era in medicine. However, it will take a long time before the Nobel Prize-winning discovery will be widely used in the treatment of a wide range of diseases.
Pioneers of RNA Interference One of the first ― back in 2002, that is, 5 years before the Nobel Prize in Medicine or Physiology was awarded to scientists who discovered RNA interference - scientists from the American pharmaceutical company Merck & Co. began to use and optimize the technology based on RNA interference for basic research., Inc.".
Their focus on transcriptional analysis (profiling) of gene expression, which is presented as a platform for the development of new drugs, contributed to the early adoption of RNA i-technologies, which offered a new and effective way to reduce the in vitro activity of a gene associated with a disease.
Gene expression is a process in which hereditary information from a gene (a sequence of DNA nucleotides) is converted into a functional product – RNA or protein. Expression can be regulated at all stages: during transcription, during translation, and at the stage of posttranslational modifications of proteins.
Dr. Alan Sachs, Vice President, Head of the Department of RNA Therapy and Molecular Profiling at Merck Research Laboratories, believes that among several factors that contributed to the progress of RNA i-technologies and the fact that they migrated from the laboratory test tube of the researcher to clinical practice, is Dr. Alan Sachs, Vice President, head of the Department of RNA Therapy and Molecular Profiling at Merck Research Laboratories, a division of "Merck &Co.", we can first of all note the end of the Human Genome project ("Human Genome Project"; www.ornl.gov ), which made it possible to create short double―spiral interfering RNAs, or small interfering RNAs - si RNAs, from the English small interfering RNAs also known as silencing RNAs (from the English. silencing ― silencing, suppressing), having the ability to "turn off", "jam" any gene, that is, to make it "inactive". Secondly, scientists have found out that many of the newly discovered targets that have been identified through gene expression, as well as through experiments on RNA interference in cell culture tissues, are likely to be inaccessible to low-molecular drugs (small-molecule drugs) or biological drugs. That is why the invention of a method of influencing these targets at the level of informational RNA (and RNA; messenger RNA ― m RNA) scientists were even more interested. And the acquisition of Merck&Co. based in San Francisco biotech company "Sirna Therapeutics Inc." (www.sirna.com ) in December 2006 supplemented the capabilities of the first in the main RNA study and provided the necessary technologies for the creation of RNA i-therapeutic agents.
Since then, a systematic, step-by-step strategy has been implemented for the integrated implementation of RNA i-technology at all stages of drug development, as well as their introduction into the research process, the design of which could allow using the full rich potential of this technology.
The goals of this strategy are to (1) identify new targets for therapeutic effects in the near future; (2) make it more likely that programs for the creation of low-molecular-weight drugs and biological drugs with properties specified at the early stages of their development will be successful.; (3) to create a reliable and widely applicable technological platform that in the long term will allow the development of drugs based on RNA interference for the treatment of a wide range of diseases.
Search for new targets The discovery, made by a lucky chance and showing that short double-stranded RNA (si RNA) can be used to activate the natural mechanism of silencing ("turning off") of genes, had a great impact on fundamental research.
RNA i-technology has made it possible to specifically inhibit the expression of any of the genes without the use of time-consuming methods, as well as methods that require a lot of time. However, in the early stages, researchers encountered obstacles in the management of gene activity in cell cultures using synthetic si RNA. For example, the creation of libraries (genome-scale libraries) of the si RNA genome required the development of algorithms that could predict the sequence of each gene in the genome of effective si RNAs (Jackson A.L., Bartz S.R., Schelter J. et al., 2003).
By 2006, the laboratory of Peter Linsley (Peter Linsley) together with Rosetta Inpharmatics LLC. (www.rii.com the selection of si RNA sequences was optimized and chemical modifications of the structures of their molecules were studied, which minimized the likelihood of "misses", incorrect effects (off-target effects), and also contributed to increasing their effectiveness (Jackson A.L., Burchard J., Schelter J. et al., 2006).
The presence of targets for si RNA exposure in the mouse and human genomes, as well as the development of an automated and powerful screening system for cell culture tissues, made it possible to conduct research on the "knockout" of the entire genome. The high performance of RNA i-screening has quickly become an important strategy in the analysis of molecular pathways. Recently published articles describe the use of RNA interference in the screening of the entire genome - from mammalian cells to drosophila ― in order to study various processes, such as Wnt protein signaling (Sepp K.J., Hong P., Lizarraga S.B. et al., 2008; Tang W., Dodge M., Gundapaneni D. et al., 2008).
The Wnt protein is a universal regulator of the individual development of animals, gives polarity and the ability to direct movement not only to the cells of the developing embryo or regenerating limb, but also to cancer cells.
In pharmaceutical research laboratories, si RNA screening is widely used to identify, confirm and select new therapeutic targets. Merck & Co. actively uses such technology, through which work is underway on those research targets in which the researchers of this company are interested. For example, in research in the field of oncology, the entire genome is screened in order to identify those genes whose exposure to chemotherapeutic agents will weaken the growth or survival of tumor cells, but at the same time this treatment will be indifferent to healthy cells. Si RNA is also used in screening targets of other diseases, including metabolic disorders, cardiovascular diseases and neurological disorders. For example, John Majercak and colleagues used this approach to analyze the metabolism of β-amyloid, which is involved in the development of Alzheimer's disease (Majercak J., Ray W.J., Espeseth A. et al., 2006).
Thanks to the latest achievements in this industry, in particular the creation of a high-performance screening model, it is now possible to systematically carry out a parallel "knockout" of thousands of gene combinations and in real time to investigate phenotypic changes taking place in a small number of cells. It is also a powerful tool for the analysis and development of integrative genomics, which has significant potential. Together with genetics, genomics and other areas of biological knowledge, modern si RNA screening can be used to detect disorders in the relationships between genes, identify and confirm important points within these systems related to the processes of disease formation (Schadt E.E., 2007). Significant investments are being made both in the development of automated screening technology and in the creation of labor-intensive bioinformatic infrastructures necessary to study in detail the mechanisms of disease development, which can also lead to the discovery of new targets for therapeutic effects in such common types of pathology as cancer, Alzheimer's disease, diabetes mellitus, obesity and atherosclerosis.
Confirmation of the correctness of the selected targets Most often, the hopes associated with the study of RNA interference focus on the prospect of creating drugs of broad therapeutic effect.
At the same time, with the help of this technology, a search is being conducted for specific targets that drugs would directly affect. The history of the development and study of monoclonal antibodies is most instructive here. Scientists Cesar Milstein and Georges Köhler studied the technique of obtaining hybridoma in 1975, but only in the 1990s drugs based on monoclonal antibodies were approved for use in patients with such common diseases as cardiovascular diseases and cancer.
Both scientists were awarded the Nobel Prize in Physiology or Medicine in 1984 "For theories regarding specificity in the development and control of the immune system and the discovery of the principle of production of monoclonal antibodies." They developed a technique for obtaining monoclonal antibodies.
Hybridoma is a hybrid cell line obtained as a result of the fusion of two types of cells: B-lymphocytes capable of forming antibodies obtained from the spleen of an immunized mouse, and myeloma cancer cells. Cell fusion is performed using a membrane-disrupting agent such as polyethylene glycol or Sendai virus. Since myeloma cancer cells are "immortal", that is, they are able to divide a large number of times, after fusion and appropriate selection, the hybridoma producing monoclonal antibodies against the antigen can be maintained for a long time.
However, by the early 1980s, the technology of obtaining monoclonal antibodies was already a revolutionary practice in immunology and pathology, contributing to the improvement of disease therapy and facilitating the solution of issues related to diagnosis and treatment. Merck &Co.'s step-by-step strategy for the development of RNA interference also includes the use of RNA i-technology to obtain valuable information that will facilitate correct decision-making at all stages of drug development and increase the likelihood of success in traditional therapy.
During preclinical studies, RNA i-technology can be applied in animal models to assess the physiological effects of "knockout" of specific target genes. Recently, viral vectors constitutively expressing active short RNAs forming "hairpins" (short hairpin RNA, or small hairpin RNA ― shRNA) have been introduced into the body of mice in order to "knock out" the expression of many target genes in vivo, including the Duchenne dystrophin gene and liver genes controlling gluconeogenesis involved in the development of type II diabetes mellitus (Ghahramani Seno M.M., Graham I.R., Athanasopoulos T. et al., 2008; Ruiz R., Witting S.R., Saxena R. et al., 2008). It is expected that the results of experiments on the "knockout" of genes using si RNA in other animal models (for example, such as primates) will provide additional information that will help make a decision whether to continue to look for new targets for exposure to low-molecular-weight drugs.
In clinical settings, by single or repeated administration of optimized si RNA to the patient, biomarkers (for example, blood glucose level or low-density lipoprotein cholesterol level) can be established, which will allow quite quickly ― in a few days or weeks ― to evaluate the physiological effect of inhibition of the target. And despite the fact that there are still some unresolved technical problems, research on si RNA with human participation may begin in the near future. Obtaining confirmation of the effectiveness of exposure to the targets selected by RNA interference in patients will facilitate making the right decision and ultimately increase the likelihood of successful use of low-molecular or biological drugs.
Measuring the chances of success Interest in treatment methods developed on the basis of RNA i-technologies began shortly after the discovery in the 1980s of enzymatically active RNAs, the so‑called ribozymes, as well as antisense oligonucleotides.
Ribozyme ―short for "ribonucleic acid" and "enzyme"), also called enzymatic RNA or catalytic RNA, is an RNA molecule that has a catalytic effect. Many ribozymes of natural origin catalyze the cleavage of themselves or other RNA molecules, in addition, the formation of a peptide bond in proteins occurs with the help of the ribosome RNA. In the framework of research on the origin of life, it was possible to create artificial ribozymes such as RNA polymerases, capable of catalyzing their own assembly under certain conditions.
The prospect of developing drugs that would be able to inhibit the activity of a particular gene at the transcriptional or post-transcriptional level has led to the emergence of many hypotheses and, as a result, the entire industry using these approaches. More than two decades later, 25 candidate drugs based on antisense RNAs began to be studied in clinical trials, but only one of them, a product for use in ophthalmology, was approved by the US Food and Drug Administration (FDA) for use in patients (Sepp-Lorenzino L., Ruddy Mk., 2008). And no means based on ribozymes has achieved success in development.
The history of the development of therapeutic approaches based on RNA, taking into account the current interest in RNA interference, makes the prospect of creating medicines based on these technologies more and more close. And along with the strategies associated with these technologies, it is important to constantly keep in mind a number of several important issues ― drug delivery to the target, selective selection of targets, as well as the determination of safety characteristics, ― before we can fully understand what the therapy based on RNA interference promises us.
Delivery Effective delivery is the single greatest barrier to the widespread adoption of therapeutic methods based on the use of RNA interference.
"Naked" nucleic acids (naked nucleic acid) ― even those that have been chemically modified to make them resistant to degradation by endogenous nucleases ― have a short half-life, are very quickly excreted from the body.
"Naked" nucleic acids (naked nucleic acid) ― a system for transferring functional genes without the participation of viruses, in which plasmid DNA can be directly transfected into cells by direct injections or injected using a so-called "gene gun" that shoots microparticles of gold/tungsten with DNA deposited on them (Wolff, J.A., Malone, R.W., Williams P. et al., 1990; Cheng L., Ziegelhoffer P.R. and Yang N.S., 1993).
As such, the administration of drugs for systemic action with unprotected si RNA is impractical. Currently, the main approach to overcoming the problems associated with a short half-life and low bioavailability of such drugs is their local administration. So, the most promising among the candidate drugs are injected directly into the target organ, for example, into the vitreous body of the eye, or applied to mucous membranes, such as the epithelium of the respiratory tract (by nasal spray or inhaler).
Effective systemic use of drugs derived from RNA interference will require the development of special platforms for the delivery of active substances. They should contribute to the stability of such drugs and their bioavailability, and should also allow molecules to reach target cells and directly penetrate them. One strategy is to encapsulate si RNA inside lipid nanoparticles (stealth liposomes), although this approach has some drawbacks. Firstly, lipid nanoparticles are not distributed throughout the body, but accumulate primarily in the liver and spleen. In addition, the use of lipid nanoparticles may be associated with toxic effects that significantly limit the use of therapeutic doses of the drug, which may become an obstacle to prescribing therapeutic concentrations of si RNA to patients (Nguyen T., Menocal E.M., Harborth J., Fruehauf J.H., 2008). Finally, the use of preparations based on lipids or polymers has certain limitations, primarily in the development of parenteral forms. In addition, despite the fact that the use of such drugs may well be suitable for the treatment of certain diseases, they will not have commercial benefits for a biopharmaceutical company, and, therefore, will not be used to create drugs for the treatment of many common diseases, such as hyperlipidemia, obesity and diabetes. Moreover, the delivery of si RNA to any of the specified cells, tissues or organs (which is often called targeted delivery), according to some scientists, may require the development of various approaches, each of which will need to be optimized for a specific organ/cell (Downward J., 2004).
Target selectionProbably, not all diseases will be equally amenable to therapy based on RNA interference, even if technical advances can take into account and ensure effective delivery of molecules to the right tissues or organs - si RNA significantly weakens gene expression, but does not "turn it off" completely, says Dr. A. Sachs.
For some types of pathology, such as cancer, "turning off" the activity of specific genes may be desirable or even necessary. For the treatment of other diseases, this approach may not be entirely sufficient, if it concerns, for example, the impact on the mechanism of disease development. Choosing the right, justified targets for exposure to such methods will require careful preclinical and early clinical study in order to present biological and clinical evidence of the validity of such an approach.
It is also necessary and important to consider whether treatment based on RNA interference has significant clinical benefits in comparison or in combination with currently available approaches and drugs. So far, the decision on the development of a particular drug has been made primarily taking into account how easy it is to ensure the local supply of a drug developed on the basis of RNA i-technology. Today, companies see their goal in identifying and developing methods of differentiated RNA interference therapy, which will significantly improve the condition of patients with common diseases when delivery of both one si RNA and their combination is provided, or the introduction of a combination of si RNA with other means, for example, low-molecular-weight drugs.
Security issues There are still several issues related to the safety of using drugs based on RNA interference technology.
For example, as in the case of drugs that are familiar to us, it is difficult to predict how the inhibition of one specific target will affect the activity of other genes.
Another thing that causes potential concern for scientists is the results of observations showing that the introduction of nucleic acids into the bloodstream can become a trigger (that is, a trigger) of a natural immune response leading to the activation of cytokines such as interferon and tumor necrosis factor. The development of this effect can be restrained or mitigated by chemical modification of the si RNA molecule, although, most likely, in each individual case it will not have the same effectiveness. Understanding the toxicity profile of systemic therapy based on RNA interference, in addition, will also be complicated by the imposition of side effects of a stealth liposome or other carrier. For example, lipid nanoparticles can activate the immune response and complement system (Sepp-Lorenzino L., Ruddy M.K., 2008; www.nature.com ). Therefore, scientists will also face the task of understanding and then minimizing the toxicity profiles of products obtained on the basis of RNA interference.
In order to solve each of these problems, an examination of the chemistry of RNA and its modifications is applied, an examination of molecular features, identification of biomarkers, preclinical safety assessment, experimental medical research and study of the design of clinical trials are carried out. For example, microarray profiling is used to evaluate off-target effects that si RNA can have in human cells, and biomarkers are also identified that will eventually be used in clinical trials involving humans in identifying various potential problems at early stages of study. And in the preclinical toxicological programs and phase I of the clinical trial, it is planned to include routine measurement of cytokine and complement activity to assess the possible effects of candidate drugs on the immune system. In addition, the researchers expect to use plasma biomarkers and the results of a molecular profiling study in order to understand the effects of si RNA at the tissue level and to find differences between toxicity caused by oligonucleotides (oligonucleotide-related toxins) and toxicity directly related to the carrier (vehicle-related toxins).
At the same time, the technology based on RNA interference is still a young field, and careful work is needed so that a viable enterprise with a long-term development perspective can be created on its basis. The successful launch of the RNA i-platform for the creation of medicines will also require significant long-term investments and will probably be characterized by step-by-step progress, rather than a one-time breakthrough that will solve all problems at once. However, as soon as all the initial obstacles are overcome, researchers will face a number of previously familiar questions, for example, how to improve those of the molecules that have proven effective.
Better and bigger and better Technologies based on the phenomenon of RNA interference, which are at the very peak, are already widely used by biotech companies today not only to solve the qualitative, but also quantitative tasks described above.
Thus, the biotech company "Alnylam Pharmaceuticals Inc.", based in Cambridge (Massachusetts, USA), is already using RNA interference technology in its research on the development of new drugs. It uses short double-stranded RNA fragments (si RNA) not only to reduce the activity of certain genes associated with diseases, but also to produce more of the necessary proteins. Currently, Alnylam Pharmaceuticals plans to use this technology in order to establish the production of medicines based on proteins ― preparations of monoclonal antibodies, vaccines and other biological products. And relatively recently, on November 12, 2009, the company announced the creation of a new enterprise called "Alnylam Biotherapeutics", which will develop this technology and cooperate with other biopharmaceutical companies interested in applying an approach aimed at the production of drugs based on cell cultures (www.alnylam.com ).
According to John Maraganore, head of Alnylam Pharmaceuticals, the company sequenced (decoded the sequence) the genome of the ovaries of the Chinese hamster ovary ― CHO, the most widely used for the synthesis of recombinant proteins after preliminary amplification (formation of additional copies of chromosomal DNA sections) of genes introduced into cells. Using the genome, Alnylam Pharmaceuticals is designing si RNAs, with the help of which it is possible to increase the lifespan of cells, which means potentially increasing the amount of synthesized protein for the production of biological drugs. For example, suppression of the expression of two genes regulating the mechanism of cell death increased cell lifespan by about 40%, and the effect on the gene involved in lactic acid metabolism – by 60%. According to the head of the company, the technology of RNA interference can be used to change the cell so that it lives longer and divides more often, and, consequently, produces more of the necessary peptides.
This approach is no longer new and has precedents. So, a group of researchers from The Singapore Bioprocessing Institute of Technology (Bioprocessing Technology Institute) was able to demonstrate that with the help of RNA interference, it is possible to more than double the number of proteins produced in the culture of CHO cells. According to one of the leaders of this project, Dr. Zhiwei Song, the main task now is to develop a technique for using RNA interference in large-scale cell cultures, for example in bioreactors.
The opportunity to increase the productivity of a cell culture without changing its qualitative composition, says Derek Ellison, chief operating officer of the British contract biotechnology company Eden Biodesign Ltd., of course, is of great interest to the biotechnology industry. However, first of all, Alnylam should prove that RNA interference does not introduce impurities or does not change the quality of the biological products synthesized in this way.
Suh-Chin Wu, a bioengineer in the field of cell cultures from National Tsing Hua University in Taiwan, who recently wrote a review on the use of RNA interference in CHO cells, believes that there will be no problems with drugs obtained using RNA interference quality and safety. Such biologics, which are currently undergoing various stages of the final phases of clinical trials, have not demonstrated any serious side effects in patients. So, the introduction of si RNA into a cell culture should not subsequently pose a threat to health or entail any problems with the approval of such drugs by regulatory authorities, he says.
However, not everyone believes in the success of this technology. And even if it turns out to be effective, it will not be financially profitable," says Tillman Gerngross, a bioengineer from Dartmouth College in Hanover, New Hampshire, USA. Production costs account for only a small fraction of the total cost of the drug. Therefore, it is more promising to work towards improving the quality and efficiency of biological products, rather than increasing quantitative production, he believes.
The Road to the Future RNA i-technology is a highly potential tool capable of revolutionizing everything related to the discovery and development of medicines: from simply identifying new targets and proving the validity of their choice to obtaining positive results and confirming their validity at earlier stages, as well as creating new ways of therapeutic effects.
The potential benefits are difficult to overestimate. They include both a significant compression of drug development schedules (see Figure) and the development of therapies for diseases that previously could not be treated. However, there are many open questions. Solving complex problems and, ultimately, a full understanding of the prospects that RNA interference carries with it will require insight, perseverance, discipline and systematic application of all scientific innovations, as well as the development of the latest drug evaluation tools, so that all this together affects the new technology.
Portal "Eternal youth" http://vechnayamolodost.ru27.01.2010