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Technology, Research and Development

CTO Kenichi Yamamura

As a R&D focused bio-venture company originating from Kumamoto University, TRANS GENIC Inc. aims to contribute to the society through the development/utilization of biological resources from individual organism to genome, and promote its business. At the beginning, the driving force for our business was the technologies of knockout mouse production and antibody production. Currently, however, in addition to genomics business, TRANS GENIC is comprised of CRO business, advanced medical business, and pathological diagnosis business for totally supporting business for drug discovery. We possess following patents for advancing these businesses: exchangeable gene trap method for the purpose of disrupting mouse (Mus musculus) gene as a model animal, GANP® mouse that produce high-affinity antibody, cancer marker in urine, and pancreatic cancer marker.

Basic research on human is becoming active in 21 century. Genome analysis technology has improved immeasurably, and disease-associated genes are revealed one after another. Can these technologies cure diseases? Unfortunately, there is always an insurmountable obstacle between gene and disease. Demonstration of cause-and-effect is extremely difficult, because human cannot be direct experimental object. Therefore, model animal as an experimental object is necessary. Human and other animals, however, are different genetically and phenotypically, so there is a compelling need for innovative technologies to overcome this problem. We consider “humanized mouse” as a solution to this problem, and strive toward an achievement by deepening traditional genetically engineered mouse-related technology.

Our platform technologies are listed below.

Genetically engineered mouse: knock-in mouse and knockout mouse

 One of the business foundations of TRANS GENIC is genetically engineered mouse production technology. Genetically engineered mouse is very important biological resource that provides new information in the research field leading to drug discovery, as well as in the basic research field, such as analysis of each gene function in individual organism, identification of relationship between gene and pathology, and pathological model mouse production. Genetically engineered mouse includes knock-in mouse and knockout mouse. Knock-in mouse is the technology for expressing gene excessively or ectopically. Knockout mouse is literally the technology to fully disrupt particular gene or to insert small mutation.

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The importance of in vivo analysis at the individual level

 The protein function can be investigated in the test tube by using cultured cells. These in vitro analysis revealed a great deal of evidence how each protein functions of its own. Many proteins, however, are intertwined and interacted with each other in the living organism. Therefore, observation at the individual level (in vivo) is needed in order to clarify how each protein acts and makes high order of life activity possible in the living body. Actually, it is said that 20-30% of knockout mice do not demonstrate phenotypic abnormality. This case does not automatically mean that the intended gene is unnecessary for the living organism, since the “fail-safe” system that other gene can complement in case when one is lost. This means that the data acquired in vitro is needed to reconfirm in vivo. TRANS GENIC has the great advantage in in vivo analysis.

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Top-level technology for knockout mouse production

Top-level technology for knockout mouse production

 Reverse genetics is an approach to analyze the function of gene and protein produced from said gene by disrupting gene encoding specific protein and determining its effect in the living organism. One of the techniques of reverse genetics is production and analysis of Knockout mouse. Model animal is needed for individual-level experiment. Especially, subhuman mammal is used in order to understand human. Among mammals, mouse is often used as an experimental animal because it is small and productive, and has a short generation time with pregnancy period of about 20 days. Moreover, according to the recent result of whole genome sequencing, it is revealed that humans share 99% of their genes with mice. Therefore, it is considered to be possible to extrapolate the role of gene in human from research result in mouse.
 “Knockout mouse” is genetically engineered mouse in which specific gene is disrupted. Knockout mouse was first produced in 1989 by the development of ES cell (Embryonic Stem cell) and homologous recombination method. ES cell is a cell line established from mouse blastocyst, which is the early–stage embryo developed from fertilized egg. It is demonstrated that blastocyst mixed with ES cell develops to a chimeric mouse that has ES cell-derived tissue. It became clear that crossing of chimeric mouse provides ES cells-derived offspring. Therefore, if genetic mutation is created in ES cell, mouse strain with this mutation can be produced. Homologous recombination is used to disrupt the target gene in ES cell. In this method, “targeting vector”, DNA that homologous region is combined on both sides of the drug-resistant gene, is constructed. When targeting vector is introduced in ES cell, recombination occurs in the homologous region between the vector and genome, and drug-resistant gene is inserted in the genome. This insertion prevents the target gene from proper transcription, results in the disruption of this unique gene. This method became popular for individual-level gene analysis after 1989. Drs. Martin Evans, Oliver Smithies, and Mario Capecchi were awarded Nobel Prize in Physiology or Medicine for developing knockout mouse production method. The actual steps involved in generating genetically engineered mouse are indicated in Fig. 1. Targeting vector is constructed by DNA cloning method based on the information in genomic database, and inserted into ES cell via electroporation. The cell that has incorporated the targeting vector into its gene is selected by screening, mixed with the embryo of wild-type mouse through aggregation method, and implanted into the recipient female mouse. The chimeric mouse is generated in this manner. The chimeric mouse is bred with a wild-type mouse, and the offspring that are capable of passing the targeting vector-derived mutation to the next generation is the knockout mouse.
 TRANS GENIC launched the contracted knockout mouse production service in 2000, has and achieved top-level production efficiency by the accumulated results in this field. Currently, we apply the technology using ES cells and homologous recombination to the production of wide variety of genetically engineered mice, such as mice that are able to generate organ-specific or stage-specific gene disruption (conditional knockout mouse) and mice that express different gene in substitution for original gene (knock-in mouse) as well as mice that specific gene is disrupted connaturally. We provide mouse production service useful to shed light on the life phenomenon based on the consultation with researchers.

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Exchangeable gene trap method with general versatility

 Another way for mouse gene disruption using ES cells is gene trapping method. Exchangeable gene trap method is the unique platform technology of TRANS GENIC (Figure 2). It is very important for living organism when, where, and how much protein is synthesized from the relevant gene. The gene expression is regulated by the region of DNA called “promoter”, located upstream of the protein-encoding region. If gene without promoter is connected downstream of certain promoter sequence artificially, protein is synthesized from the gene. On the other hand, protein-encoding genetic information needs termination point. A sequence called poly A plays such role. Information downstream of the poly A is not translated, so protein synthesis terminates. In exchangeable gene trap method, exchangeable trap vector that has drug-resistant gene without promoter sequence but with poly A-containing sequence is introduced to ES cell. Exchangeable trap vector is inserted randomly into the ES cell genome. If the insertion site is located in the first region of protein-encoding DNA, protein synthesis from genetic region downstream of the insertion site is inhibited by poly A, results in gene-disrupted cell incapable of synthesizing this protein. Inserted gene is identifiable based on the known sequence of exchangeable trap vector. Also, large-scale production of gene-disrupted mouse is possible by disrupting gene randomly. Moreover, since the exchangeable trap vector carries a mechanism for replacing gene, conditional allele can be easily produced by replacing mouse gene with human gene or inserting loxP.
 TRANS GENIC provides TG Resource Bank, the series of genetically engineered mouse produced through the above unique exchangeable gene trap method. This genetically engineered mouse library is retrievable and available to purchase for any individuals. New disease model mouse such as obesity model mouse (as described below) is discovered from this library, and expected to be a useful life resource through phenotyping.

Exchangeable gene trap method with general versatility
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CRISPR/Cas9 technology: faster and more high-efficient production of genetically engineered mouse

Genome editing(CRISPR/Cas9) technology: faster and more high-efficient production of genetically engineered mouse

 In recent years, genome editing technology has moved into the limelight as a novel genetic modification technology. Genome means the entire genes included in the cell. Genome editing technology is to edit the genome at arbitrary place and to modify the gene information existing in this place.
 In this method, genome is cleaved in any place using genome-cleavage enzyme. Then, cell tries to restore the cleaved genome. This cleaved part can be modified using the error occurring during the restoration process. The current most popular genome editing technology is CRISPR/Cas9 system. CRISPR/Cas9 system utilizes the acquired immune mechanism of bacteria. Using this mechanism, bacteria recognize and cleave the genome of extraneous enemy with the enzyme called Cas9. Artificial application of this recognition/cleavage system to the cell genome enables genetic engineering. As indicated in Figure 3, guide RNA (gRNA), which recognizes DNA sequence of target genome regions is generated. When gRNA and Cas9 are introduced together into the cell, Cas9 binds to the genome region recognized by gRNA and cleaves the genome. The cleaved genome is repaired through the restorative ability called non-homologous end joining (NHEJ), however deletion and insertion will occur (Figure 3, left). As a result, mouse in which gene information of that part is modified can be produced. If donor DNA with arbitrary mutation, which shares homologous region with the cleaved genome, is added, since the DNA repair occurs under the information of donor DNA, this arbitrary mutation is introducible into the genome (Figure 3, right). In 2013, it is reported that knockout mouse can be produced by injecting target genome DNA into the fertilized egg using CRISPR/Cas9 (Figure 3, below). In this method, knockout mouse can be produced more conveniently and in one generation less (about 3 month) than conventional process without constructing targeting vector or using ES cell. Also, it is revealed that the efficiency of homologous recombination in ES cell is dramatically improved, and it enables genetic disruption that was impossible before. TRANS GENIC is licensed from the Broad Institute that developed genome editing technology using CRISPR/Cas9, and launched contract genetically engineered mouse production service using CRISPR/Cas9. Not only producing knockout mouse using CRISPR/Cas9, we are willing to try wide variety of ideas for producing novel gene modified mouse, such as introducing 1-base mutation associated with human pathological condition, and multi-site mutagenesis. By introducing this technology, the strategies previously-impossible or extremely difficult are becoming a reality, and expanding the possibility for the production of genetically engineered animals as a life resource that provide new information.

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Disease model mouse: application to therapeutic development

 Production and analysis of genetically engineered mouse is the essential way to analyze genetic function today. Especially, function analysis of human disease-associated gene by using mouse has very important role to clarify the pathgenetic mechanism. Animals that show symptoms similar to human disease can be used as disease-model mouse in order to develop therapeutic approach or preventive method. For example, we provide disease or pathological condition model mouse, such as breast cancer, obesity, and sleep disorders. Research using these model mice enables to analyze how each disease or pathological condition occurs, and how they can be prevented. Many human diseases are not clarified their pathological mechanism yet, and do not have evidence-based treatment. We will develop and introduce new kinds of disease model mouse in order to help to overcome diseases and contribute to the realization of healthy and affluent lives for people.

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Pathological condition-visualized mouse: pathological observation in the living state

 Anatomy is required to analyze a mouse with disease. In recent years, however, new technology that enables the quantitative observation in living mouse has been developed, called pathological condition-visualized mouse (Figure 5). In this model mouse, genetically modified mouse production technology is used to introduce the mechanism activating luciferase under the pathological condition. Luciferase catalyzes an enzymatic reaction of firefly luminescence, reacting with luciferin (substrate) to emit light. When luciferin is administered to a mouse with the pathological condition and producing luciferase in the body, luminescence can be observed in the pathological site. The luminescence can be detected by highly-sensitive CCD camera. This method is non-invasive, because pathological condition progressing in the living mouse body can be detected through luminescence. Also, it is possible to observe the same mouse in a time-dependent manner, repeatedly, and on the long-term basis. We provide pathological condition-visualized mouse for visualizing cellular stress and inflammation. Visualizing technology is relatively new, and considered to become popular as new animal model in the future.

Pathological condition-visualized mouse: pathological observation in the living state
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GANP mouse: antibody with high-affinity and high-specificity

 Antibody is the protein produced as one of the bio-defense system (immunity) to recognize and defend from foreign invasion or exogenous material. Antibody has remarkable recognition capability naturally (affinity and specificity for particular molecule), and used in research or diagnosis to recognize particular biomolecules. In the living body, however, there exist small quantities of protein expressed only in cancer cell or protein modified specifically in tumor. Therefore, high-specificity and high-affinity is required for antibody to be utilized as a tumor marker. GANP mouse is a technology to enhance the specificity and affinity of antibody. GANP (Germinal center-associated Nuclear Protein) is the protein expressed in antibody-producing cells. It generates somatic mutation in antibody gene by binding with AID and being carried into nucleus, consequently enhancing the specificity and affinity of antibody. GANP mouse is produced to express higher amount of GANP through genetic engineering technology. Expression level of mRNA in GANP mouse is regarded about 1.8 times as much as the normal one, and enhances to introduce more mutation in antibody gene. In consequence, probability to gain high-affinity and high-specificity antibody will be increased. We created antibodies against the candidate substances of new cancer markers through antibody production technology using GANP mouse. In the case of diacetylspermine, a protein with a molecular weight of 360, has acetyl groups at both ends, we succeeded to develop an antibody recognizing acetylspermine that has acetyl group at only one end. Also, an antibody that can recognize the hydroxylated-proline residue of a-fibrinogen was successfully developed. In recent years, these antibodies are applied to not only research/diagnostic use, but also antibody-drug. Various antibodies can be generated through GANP mouse technology even if impossible with other methods. The importance of GANP mouse technology is expected to increase in wide variety of field, from basic research to drug discovery and diagnosis.

GANP mouse: antibody with high-affinity and high-specificity
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Ultimate model: humanized mouse

 Human and mouse share almost the same kinds of gene, however generate protein with different amino acid sequence. Many proteins fulfill their function by interacting with other proteins. Therefore, a mouse introduced the human disease-causing gene does not always exhibit intended phenotype. Drug metabolism of human is extremely different from that of mouse. In the liver, the expression amount of gene and amino-acid sequence of protein between human and mouse are different: they are close but not the same. Is it possible to overcome the limitation of mouse model? How is it possible? The answer is the humanization at genetic level, cellular level, and tissue/organ level. For the humanization at genetic level, mouse gene can be replaced to human gene by using exchangeable recombinant method, patented by TRANS GENIC. CRISPR/Cas9 method is also be applied. However, humanization of every single gene is a hard work, therefore humanization of whole organ is required. It is possible to generate ideal disease model by using the technology that humanizes only desired organ. This organ-humanized mouse enables to analyze the cause of disease and pathological condition, and even applicable to the development of treatment method. We are engaged in the development of ①inducing differentiated cell from human iPS cell, ②the recipient mouse in order to transplant to ①, ③organ-humanized mouse using ① and ② (Figure 6).

Ultimate model: humanized mouse
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