Development of technology for inducing the gene silencing with near infra-red light

Transcription

1 No. 200 Development of technology for inducing the gene silencing with near infra-red light Takashi Ohtsuki Professor Graduate School of Natural Science and Technology 1. Summary of the research The researchers have recently developed technology to induce the gene silencing through irradiation with near-infrared light. This technology can be applied to medical treatments and in the analysis of gene functions. There has been no technology developed until now for inducing the gene silencing with near-infrared light, which penetrates body tissue well, and it is expected that this will be developed into a treatment technology that has extremely small negative effects on normal cells. The objective of this research is to:(a)increase the gene expression inhibition rate of this technology,(b)expand the range of near-infrared wavelengths that can be used and(c) demonstrate applications of this technology for medical treatment. 2. Superiority of the developed technology The technology in patent application is a technology that makes it possible to deliver RNA into cells using near-infrared light at the times and in the locations of irradiation. In particular, the purpose of this technology for this application is causing the gene expression to be interfered by the RNA delivered into the cell. This mechanism is based on the features of a carrier molecule developed by the applicants. The carrier molecule is a molecule consisting of a photosensitizer part and a protein part which delivers the RNA into the cell. Although RNA can be delivered into cells by the carrier molecule, it becomes trapped in small vesicles known as endosomes where it cannot function at all. However, by exposing the cell to the excitation light that activates the photosensitizer, it is possible to free the RNA from the endosome(fig. 1, left). Initially, the only light that could be used in this technology was visible light, but recently, through testing of several types of photosensitizers, it has become possible to use near-infrared light, which penetrates body tissue well. As a result of this progress in the technology, it has become possible to deliver RNA into cells and induce RNA function by irradiating the cell with near-infrared light. There is an advanced technology to induce RNA function using light and caged RNA. Caged RNA is RNA with a protective base that can be removed by light. Caged RNA is inactive until it

2 is exposed to light, after which it becomes active. However, the light used to remove the protective base from caged RNA is primarily light with wavelengths in the ultraviolet range. There have been no reports of examples that use near-infrared light, which easily penetrates deep into the body. Also, when using caged RNA, there needs to be an accompanying technology for introducing the RNA into the cytoplasm. The present technology, which regulates the introduction of RNA into the cellular cytoplasm using near-infrared light, is superior to caged RNA technology in these points. In patent application , patents are being applied for the candidates for the [protein part] and [photosensitizer part] that comprise the carrier molecule as well as the types and uses of the [RNA that can be delivered] by the carrier molecule. Of the examples of RNA delivered, the most important is short hairpin RNA(shRNA). By delivering shrna into the cell, it is possible to [interfere the expression of genes with DNA sequences that correspond to the shrna sequence](a phenomenon referred to as RNAi). In other words, this is a technology that interferes expression of a specific gene in cells irradiated with near-infrared light(fig. 1, right). Fig 1 (left)the carrier molecule developed by the applicants and the mechanism for the deliver of RNA into the cell by irradiation with light. (Right)An example in which the genes are interfered only in the cells exposed to light. In this example, shrna that causes silencing of the GFP gene was used, and the gene silencing was evaluated using the green fluorescence of GFP as an indicator. 3. Marketability and future of the technology The invented carrier molecule in patent application can deliver any specific RNA that can bond to the carrier molecule into the cellular cytoplasm. Therefore, this technology can be utilized in treatment techniques that introduce RNA that has a therapeutic effect on a disease into cells as well as in research methods for investigating the intracellular functions of RNA. This technology may be released in the market as a carrier molecule/rna composite pharmaceutical(as a set with a light emitting device) or a research-use reagent kit that contains the carrier molecule

3 The RNA that can be delivered into the cell by the carrier molecule may be RNA with any of a variety of functions(interfering gene expression, promoting translation, splicing, bonding to particular molecules, etc.)of these types of RNA, shrna has a high level of applicability. It is well-known that shrna can be freely designed to interfere expression of any specific gene. For example, it is possible to artificially design and synthesize shrna that can interfere the expression of disease causing genes, which can lead to treatments for such diseases. In addition, microrna(mirna)has received a lot of attention recently as a natural RNA that has the ability to interfere gene expression, and mirna has been shown to be related to diseases. The major feature of this technology is that is can interfere gene expression only in cells that are irradiated with near-infrared light. Because near-infrared light is penetrates body tissue well and the exposure does not damage cells or cause heating, it is the most convenient light that can be used for locations deep within the body. In addition, the carrier molecule used in this research has a small photosensitizer functional base but the majority of the molecule is protein, so it is known to be biodegradable and has a low level of cellular toxicity. Based on the above explanation, it is expected that this technology will develop as a therapy technology that has extremely small negative effects on normal cells. 4. Objective and necessity of the research In this experimental research, the technology stated in patent application (technology for delivering RNA into the cytoplasm and inducing the RNA function by irradiating the cell with the near-infrared light)will be studied as [a technology for inducing the gene silencing with near-infrared light.] Although the practicality of this technology as [a technology for inducing the repression of gene expression with near-infrared light] has been demonstrated, the problems with the research results to date are that:(a)an 80% RNAi efficiency has not been achieved,(b)the nearinfrared light that can currently be used is limited to near 750nm and(c)there has been a lack of actual applications to medical treatments, such as the repression of cancerous cell growth. Research will be done for the purpose of resolving these issues. 5. Methods and techniques of the research The research team representative will perform the following research in order to demonstrate practicality as a technology for inducing the gene silencing with near-infrared light. 1)Optimize the method with the aim of achieving at least a 90% RNAi efficiency Currently the RNAi efficiency(in the standard GFP inhibition test used by the applicants)is about 70%, which is believed to be insufficient for practical use. In other words, although expression is inhibited, 30% of the genes are still being expressed, which makes it difficult to repress the growth of cancer cells. Given this, the aim is to achieve a 90% RNAi efficiency by considering various conditions. Our goal is that the expression of the target gene will be 10% or less, which opens the way towards applications for repressing cancer cells. 2)Expand the usable range of near-infrared wavelengths

4 Presently the only photosensitizer that can be used in this technology is a near-infrared fluorescent pigment with maximum absorbance at 750nm. However, for lasers, a photosensitizer that absorbs wavelengths around 785nm and 685nm would be more versatile. Therefore, by making it possible to use near-infrared light around those wavelengths, the versatility of this technology would increase. In order to increase the versatility of the technology in this study, the range of usable wavelengths will be increased through study of various types of photosensitizers in the carrier molecule. 3)Repress the growth of cancer cells An actual application of this technology(technology for inducing the gene silencing with near-infrared light)will be demonstrated, one which makes it possible to repress the growth of human cancer cells. Either SW480 human colon cancer cells or 211H human malignant mesothelioma cells will be used. Examples of the shrna will include one that recognizes the mutation at the 12th position of the K-Ras gene codon in SW480. Also, various shrna sequences that target expressed genes unique to cancer cells will be tested. From those tests, an shrna that represses cancer cell growth merely by exposure to near-infrared light will be obtained. Through the above research, the usefulness and versatility of the technology for inducing the gene silencing with near-infrared light will be increased, and actual examples that can lead to the transfer of the technology will be shown. 6. Objectives of the research Objective 1: Achieve at least a 90% RNAi efficiency Aim for the improvement of the inhibition rate to at least 90% by incubating the cells in the presence of the carrier molecule and shrna under optimal conditions, and by optimizing the strength of irradiation. Objective 2: Expand the usable range of near-infrared wavelengths Through study of the various types of photosensitizers that can be used in the carrier molecule, expand the range of near-infrared wavelengths usable with the technology to about nm. Objective 3: Repress the growth of cancer cells By using this technology to inhibit the expression of genes unique to cancer cells, show actual examples where it is possible to repress the growth of human cancer cells

5 7. Target patents Patent name Patent application number Applicant Inventors Summary New carrier molecule and method for delivering RNA into cells using near-infrared light Patent application Date of the application 2011/03/ Takashi Ohtsuki, Yuka Ishiodori [Topic] Provide a technique that uses near-infrared light(preferably with a wavelength between about nm), which does not cause cellular damage or heating and penetrates body tissue well, as the means of introducing RNA into cells. [Solution] Carrier molecule is characterized by a structure consisting of a carrier protein containing a cell membrane penetrative peptide(cpp) and an RNA bonding protein(rbp), and a photosensitizer(pst)that functions when exposed to light in wavelengths in the near-infrared region, and that is bonded to the N terminal or C terminal of the carrier protein. A carrier molecule/rna composite is formed consisting of the carrier molecule and RNA bonded to the RBP of the carrier molecule. The composite is put in contact with the cell and made to enter an endosome inside the cell. Then, the cytoplasm is irradiated with nearinfra red light causing the PST in the carrier molecule/rna composite to function, dispersing the composite in the cytoplasm at which point it is possible for the function of the introduced RNA to be induced. Contact Details Yutaka Watanabe, Ph.D. Manager Intellectual Property Office Organization for Research Promotion and Collaboration Phone: wyutaka@cc.okayama-u.ac.jp Web Site: