생명과학회지 (Journal of Life Science)

  • 제22권6호
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  • Pages.703-712
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  • 2012
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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다양한 adeno-associated virus (AAV) 혈청형의 효율성 높은 생산법과 새로운 공통적 정량법 개발

Improved Production Efficiencies of Various Adeno-Associated Virus (AAV) Serotypes and a Novel Universal AAV Titration Method

  • 조영화 (주성대학교 주성유전자치료기술센터) ;
  • 최예진 (식품의약품안전평가원 생물의약품연구과) ;
  • 윤정희 (주성대학교 주성유전자치료기술센터) ;
  • 김남희 (식품의약품안전평가원 생물의약품연구과) ;
  • 최미라 (식품의약품안전평가원 생물의약품연구과) ;
  • 최영국 (주성대학교 주성유전자치료기술센터) ;
  • 김경희 (주성대학교 주성유전자치료기술센터) ;
  • 이영일 (수원대학교 공과대) ;
  • 이범준 (충북대학교 수의과대학) ;
  • 박기랑 (주성대학교 주성유전자치료기술센터)
  • Cho, Young-Hwa (Juseong Gene Therapy R&D Center, Juseong University) ;
  • Choi, Ye-Jin (Biologics Research Division, National Institute of Food and Drug Safety Evaluation, KFDA) ;
  • Yun, Jung-Hee (Juseong Gene Therapy R&D Center, Juseong University) ;
  • Kim, Nam-Hee (Biologics Research Division, National Institute of Food and Drug Safety Evaluation, KFDA) ;
  • Choi, Mi-Ra (Biologics Research Division, National Institute of Food and Drug Safety Evaluation, KFDA) ;
  • Choi, Young-Kook (Juseong Gene Therapy R&D Center, Juseong University) ;
  • Kim, Kyung-Hee (Juseong Gene Therapy R&D Center, Juseong University) ;
  • Lee, Young-Ill (School of Engineering, University of Suwon) ;
  • Lee, Beom-Jun (College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University) ;
  • Park, Kee-Rang (Juseong Gene Therapy R&D Center, Juseong University)
  • 투고 : 2012.03.20
  • 심사 : 2012.05.15
  • 발행 : 2012.06.30
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초록

AAV는 매우 안전하고 효율적인 유전자전달방법으로 인정되어 왔다. 그러나, AAV가 가진 생물의약품으로서 단점은 효율적이고 재현성 높은 생산법이 취약하고, 또한 다양한 혈청형을 간단한 한 가지 공통적 방법으로 신뢰성 있게 정량하는 방법이 개발되어야 하는 것이다. 따라서, 본 연구에서는 AAV2와 아데노바이러스를 동시에 감염하는 종래의 생산법에 의한 효율성과 새로운 생산법, 즉 AAV2 감염 후 pHelper 플라스미드를 transfection 하는 방법을 통한 생산효율성을 비교하였고, HEK293과 293T를 생산세포주로 하여 시간에 따른 생산효율성도 분석하였다. 그 결과 AAV2와 pHelper DNA를 포함한 새로운 생산법은 기존의 방법에 비해 10배 이상 높은 생산효율성을 보였고, 293T에서 AAV2를 10 MOI로 감염한 후 5일째에 가장 높은 생산효율성을 보였는데, 생산세포 한 개 당 $1.61{\times}10^5$ virus genomes (v.g.)을 생산하는 결과였다. 따라서 이 생산조건을 다른 혈청형 생산에 적용한 결과, 모든 혈청형에서 생산세포주 한 개 당 $10^5$ v.g. 이상을 생산하는 효율성을 보였다. 한편, 다양한 AAV 혈청형을 한 가지 공통적인 방법으로 정량하기 위해 the universal PCR 프라이머를 제작하였고, 그것을 이용하여 신뢰성 높고 10개 분자까지도 증폭이 가능한 결과를 모든 혈청형에서 얻었다. 그러므로 이 한 쌍의 정량용 the universal 프라이머는 임상시험용 아데노바이러스벡터에 존재하는 AAV오염을 검출하는 것에도 사용 가능하다.

Adeno-associated virus (AAV) has been considered to be a very safe and efficient gene delivery system. However, the major obstacles to therapeutic usage of AAV have been to achieve highly efficient and reproducible production processes, and also to develop a reliable quantifying method of various serotypes with a simple protocol. We compared the efficiency of the conventional production protocol of AAV2 and adenovirus (Ad) co-infection to that of a new method containing AAV2 infection followed by pHelper transfection. We tested HEK293 and 293T, and further examined the time-dependent changes of AAV2 production. The new method of AAV2 and pHelper DNA gave about ten times higher production efficiency than that of the conventional protocol. The highest production efficiency in 293T was achieved as $1.61{\times}10^5$ virus genomes (v.g.)/cell by the new method of 10 MOI of AAV2 infection and 5 days post-infection. This protocol of the highest efficiency was then applied to produce various AAV serotypes and showed the efficiencies higher than $10^5$ v.g./cell. Next, we designed the universal PCR primers of highly conserved regions for various AAV serotypes to develop a simple and reliable titration method. The universal primers could amplify all the tested AAV serotypes with similar sensitivities by ten molecular copies. Therefore, this pair of universal primers can be further utilized to detect AAV contaminants in therapeutic adenoviral vectors.

키워드

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