10: The Front Line in Gene Therapy (The Department of Molecular Genetics)

Professor Takashi Okada of the department of Biochemistry and Molecular Biology (Molecular Genetics) has been continuously carrying out research into the development of fundamental gene therapy techniques. His main research theme is the development and clinical application of genetic cell therapies using "viral vectors" for illnesses where fundamental therapies are still difficult, such as cancer and muscular dystrophy. Let's take a closer, topic-based look at the research pursued by Prof. Okada to present.

Development of new hybrid vectors
One of the research topics Prof. Okada has grappled with is the development of "hybrid vectors", which are a combination of several vectors such as retrovirus vectors and adenovirus vectors, that capitalizes on each of their strengths, and compensates for their shortcomings . "We developed hybrid vectors having a frame with the properties of an adenovirus and equipped with the innards of a retrovirus to compensate for each of their weaknesses. Using this vector, it became possible to obtain an efficient increase in expression of genes necessary for a treatment that was stable long-term, regardless of cell division" says Prof. Okada 1). For the development of this new viral vector, Prof. Okada was awarded the Japanese Cancer Association Incitement Award in 2005.

Standardization of large-volume AAV vectors
Additionally, techniques related to different viral vectors that Prof. Okada has engaged with include research into the implementation of adeno-associated virus (AAV) vectors. At present, AAVs are highly regarded as the most prominent candidate vectors for genetic therapy; however, Prof. Okada told us that "The greatest challenge in the full-scale rollout of AAV vectors was that large-scale production methods for vectors standardized by production management standards, as part of the formulation of GMP (Good Manufacturing Practice), had not been established."
By overcoming various technical challenges, Prof. Okada succeeded in developing original methods for the production of high purity and highly standardized AAV vectors 2). According to Prof. Okada, this means that "At present, the license transfer of the techniques to the bio-industry in Japan is underway, and the large-scale production of GMP preparations is being executed at viral vector production plants with a view towards clinical application". The results of this research show the state of steady progress from fundamental research to clinical application towards a full-scale rollout of gene therapies.

Vector-producing MSCs
In addition to vector related researches, Prof. Okada has also been conducting researches using stem cells called MSCs (mesenchymal stromal cells) to develop genetic therapy methods for cancer.
MSCs have the property that they chase cancer cells in the body. Consequently, if MSCs are used as "viral vector-producing cells", no matter where the cancer cells move, the MSCs will rapidly find and accumulate at the invisible cancer cells and produce the viral vector there. The method administers "cells that produce virus" into the body, instead of administering virus directly into the body3).
Regarding the advantages of utilizing MSCs in gene therapies, Prof. Okada gave us the following statement: "Firstly, by using the hunting effect of MSCs, the viral vector can be sent into permeating lesions such as those found in cancer. As a result, it is possible to reduce side effects and increase the concentration of the expression of therapeutic genes at the target tissue, as well as enabling the long-term, stable replenishment of protein within the lesion". It is hoped that, in the future, MSC gene therapies will play a role in patients where invasion or metastasis of cancer has made surgery impossible.

Gene therapies for muscular dystrophy
Whilst researching cancer therapies, Prof. Okada has also engaged in research into the development of gene therapies for genetic muscle disorders called "muscular dystrophies" that present with progressive muscle weakness and muscle atrophy. Duchenne muscular dystrophy (DMD) has a particularly high incidence rate and serious clinical symptoms; however, no effective treatment exists. However, the actual causative gene for DMD was identified as many as 20 years ago, and it is known that its occurrence results from abnormalities in the "dystrophin gene" on the X chromosome.
To date, Prof. Okada has demonstrated the efficacy of gene treatments in various DMD model animals by the introduction of the micro-dystrophin gene using the previously mentioned AAV vectors .

For example, to investigate the details of the immune response accompanying AAV vector treatment, Prof. Okada has performed experiments in dog models, which have an immune response system more similar to that of humans than that of mice 4). Prof Okada explained that, as a result, "Even in the dog model, it was observed that no inflammatory reaction to the expression of the stabilized micro-dystrophin gene occurred over 8 weeks, and we were able to reconfirm the possibility of using AAV vectors for DMD gene therapies." And additionally that "Upon using infra-red monitors to observe the frequency of movement of the treated dogs in their cages, a clear increase in the values was found compared to before the therapy. Lung and heart function also improved." Or, in other words, it could be said, "the fact that dogs have a similar viral clearance function to humans, lends considerable hope to use in humans" (Prof. Okada).

Future research and development
To conclude, Prof. Okada told us the following: "I would like to continue the research carried out to present into fundamental vector techniques, stem cell techniques, molecular pathology analysis, and the gene therapies that make use of these. Additionally, so that these results can make it to clinical use, I would like to guide the standardization of drug therapies and preparation of clinical trials".

1) Okada T, et al. In situ generation of pseudotyped retroviral progeny by adenovirus-mediated transduction of tumor cells enhances the killing effect of HSV-tk suicide gene therapy in vitro and in vivo. J Gene Med. 2004 Mar; 6(3): 288-99.
2) Okada T, et al. Scalable purification of adeno-associated virus serotype 1 (AAV1) and AAV8 vectors, using dual ion-exchange adsorptive membranes. Hum Gene Ther. 2009 Sep; 20(9): 1013-21.
3) Uchibori R, Okada T, et al. Retroviral vector-producing mesenchymal stem cells for targeted suicide cancer gene therapy. J Gene Med. 2009 May; 11(5): 373-81.
4) Koo T, Okada T, et al. Long-term functional adeno-associated virus-microdystrophin expression in the dystrophic CXMDj dog. J Gene Med. 2011 Sep; 13(9): 497-506.

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