Title:
Development of New Viral Vectors as Tools for Effective Gene Therapy

Shuji Kubo, M.D., Ph.D.
Assistant Professor
Laboratory of Host Defenses
Institute for Advanced Medical Sciences
Hyogo College of Medicine

Abstract:
　Gene therapy involves the transfer of DNA or RNA in order to modify or correct cellular functions. In the past 20 years since the first clinical trial of gene therapy was initiated, the use of modified viruses such as adenovirus and retrovirus as gene delivery vehicles (“vectors”) has become a reliable technology for cell transduction in vitro . Such vectors represent highly useful research tools, and some promising results have been obtained in gene therapy clinical trials involving ex vivo gene transfer, or requiring only low levels or transient duration of transduction. Accordingly, we have worked to develop the most high-yield, cost-effective, time- and labor-efficient processes for propagation and production of virus vectors 1-3 .

However, the generally low efficiency of direct in vivo transduction with conventional gene transfer vectors remains a significant obstacle for the field of gene therapy 4 . Therefore, to overcome this obstacle, we are further pursuing the development of novel types of viral vectors, which may improve the efficiency and effectiveness of gene transfer.

For gene therapy of cancer, which requires efficient gene delivery to eradicate tumor cells , we have developed replicating viruses (oncolytic viruses) based on adenovirus 5 or retrovirus. These oncolytic viruses are able to efficiently replicate and spread in multiple established cancer cell lines. Tumor-selective spread of these viruses was observed to mediate efficient killing of cancer cells in vitro , and also achieved significant anti-tumor effects in mouse xenograft models of human cancers.

For gene therapy of hereditary diseases and other applications which require long-term gene expression, we have also developed various hybrid virus vector systems, which employ adenovirus as a first-stage delivery vehicle to efficiently enter target cells, but then utilize the machinery of integrating viruses 6,7 or mobile genetic elements 8 to achieve chromosomal integration. After infection with these hybrid vectors, stable and long-term transgene expression through permanent chromosomal integration was observed both in cell lines in vitro and in animal models in vivo .

We anticipate that further development of innovative vector technologies and improved manufacturing methods are necessary steps that will contribute to the success of human gene therapy.

Reference:
1. Sato M, Suzuki S, Kubo S , Mitani K. Replication and packaging of helper-dependent adenoviral vectors. Gene Ther 2002; 9: 472-476.

2. Kubo S , Saeki Y, Antonio Chiocca E, Mitani K. An HSV amplicon-based helper system for helper-dependent adenoviral vectors. Biochem Biophys Res Commun 2003; 307: 826-830.

3. Yamada K, Morishita N, Katsuda T, Kubo S , Gotoh A, Yamaji H. Adenovirus vector production using low-multiplicity infection of 293 cells. Cytotechnology 2009; 59: 153-160.

4. Mitani K, Kubo S . Adenovirus as an integrating vector. Curr Gene Ther 2002; 2: 135-144.

5. Terao S, Shirakawa T, Kubo S , Bishunu A, Lee SJ, Goda K et al . Midkine promoter-based conditionally replicative adenovirus for targeting midkine-expressing human bladder cancer model.Urology 2007; 70: 1009-1013.

6. Kubo S , Mitani K. A new hybrid system capable of efficient lentiviral vector production and stable gene transfer mediated by a single helper-dependent adenoviral vector. J Virol 2003; 77:2964-2971.

7. Kubo S , Kataoka M, Tateno C, Yoshizato K, Kawasaki Y, Kimura T et al . In vivo stable transduction of humanized liver tissue in chimeric mice via high-capacity adenovirus-lentivirus hybrid vector. Hum Gene Ther 2010; 21: 40-50.

8. Kubo S , Seleme MC, Soifer HS, Perez JL, Moran JV, Kazazian HH, Jr. et al . L1 retrotransposition in nondividing and primary human somatic cells. Proc Natl Acad Sci U S A 2006;103: 8036-8041.