마스터세포은행 제조를 위한 HSV1-TK와 yeast CD 레트로바이러스 생산 세포주 구축

다국어 초록 (Multilingual Abstract)

Retroviral replicating vectors (RRVs) have shown efficient tumor transduction and enhanced therapeutic benefits in a variety of cancer models. In the previous study, we developed the spRRV system (sRRVgp-yCD & spRRVe-TK) encoding HSV1-TK and human codon-optimized yeast CD genes as for cancer therapeutic agents, respectively. In the present study, to achieve a successful clinical trial using RRVs, we were willing to establish the virus producer cells (VPCs) that produce high-titer spRRVs. First, each of the spRRVs (sRRVgp-yCD8 & spRRVe-TK) transduced separately into retrovirus packaging cell line PG13. And then, PG13/env-HSV1-TK and PG13/gagpol-yCD8 VPCs were selected clonally and amplified for the screening of VPCs producing high-titer retroviral vector particles. The characteristics of established VPCs were accomplished by validation of genome stability, infectivity, cytotoxicity, productivity, and mycoplasma test. Finally, the validated PG13/env-HSV1-TK and PG13/gagpol-yCD8 VPCs were manufactured in the GMP facility. Also, the MCBs can be used for mass production and purification of retroviral vectors in GLP or GMP facilities for non-clinical and clinical trials.

목차 (Table of Contents)

• List of Tables ·····································································································Ⅲ
• List of Figures ····································································································Ⅳ
• List of Abbreviations ···························································································Ⅵ
• Ⅰ. 서 론 ············································································································1
• Ⅱ. 재료 및 방법 ··································································································5
• 2.1. 세포 배양 ······························································································5
• 2.2. 레트로바이러스 생산 ··············································································5
• 2.3. 바이러스 physical titer 측정 ····································································6
• 2.4. 단일 세포 선별 ·······················································································7
• 2.5. 염색체 내 바이러스 벡터 integrity 확인 ····················································9
• 2.6. 마이코플라즈마 부정 시험 ······································································10
• 2.7. 바이러스 생산 세포주 (VPC)를 이용한 레트로바이러스 생산 ·······················11
• 2.8. 바이러스 biological titer 측정 ································································11
• 2.9. 전구약물에 의한 세포의 약물 감수성 평가 ··············································13
• 2.10. spRRV 바이러스 벡터 게놈 (genome) 안정성 평가 ···································13
• 2.11. 통계학적 분석 ·····················································································14
• Ⅲ. 결 과 ··········································································································15
• 3.1. 레트로바이러스 생산 ·············································································15
• 3.2. PG13 세포주 기반 spRRVe-sEF1α-TK RV 또는 sRRVgp-sEF1α-yCD8 RV생산 VPCs 1차 구축 ···························································································19
• 3.3. 레트로바이러스 벡터의 integrity와 약물 감수성 평가 ································23
• 3.4. PG13 세포주 기반 spRRVe-sEF1α-TK RV 또는 sRRVgp-sEF1α-yCD8 RV생산 VPCs 2차 구축 ···························································································27
• 3.5. 2차 구축 바이러스 생산 세포주 밸리데이션 ···············································31
• 3.6. env-TK RV와 gagpol-yCD8 RV의 생산성과 감염 효율 평가·························39
• Ⅳ. 결론 및 고찰 ································································································42
• Ⅴ. 참고문헌 ······································································································47
• ABSTRACT ········································································································51
• 감사의 글 ··········································································································53