用CRISPR/Cas9 RNPs预装的不同复原病毒样粒子的深度表征和比较
Max Wichmann1, Cecile L Maire2, Niklas Nuppenau1
1Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany.
International journal of molecular sciences
|July 29, 2023
概括
增强的复原病毒样粒子 (egRVLP) 在CRISPR/Cas基因编辑交付方面表现出卓越的效率. 这些先进的VLP提供了高淘汰率,最小的目标外影响,为更安全的临床应用铺平了道路.
科学领域:
- 生物技术是生物技术.
- 基因编辑 基因编辑
- 分子生物学分子生物学
背景情况:
- 克里斯普尔/卡斯基因编辑具有治疗前景,但需要安全高效的传递载体.
- 临床翻译受到CRISPR/Cas组件短期表达载体的缺乏限制.
- 病毒样粒子 (VLP) 正在成为CRISPR/Cas技术的潜在交付系统.
研究的目的:
- 描述和比较三种基于逆转录病毒的VLP (gRVLP,egRVLP,LVLP) 用于CRISPR/Cas传递.
- 评估这些VLP在基因编辑应用中的效率和特异性.
- 建立可靠的VLP表征和验证方法.
主要方法:
- 优化生产和统一的RVLP特征测定试验 (NTA,IFC,Cas9-ELISA).
- 对gRVLP,egRVLP和LVLP进行基因编辑293T细胞的比较测试.
- 评估人类诱导的多能干细胞 (hiPSCs) 针对CCR5.5的上位和离位编辑.
主要成果:
- 增强的gRVLP (egRVLP) 证明了最有效的基因编辑.
- 由于增强的Cas9货物转移和/或释放,egRVLP的性能优越.
- egRVLPs在高PSC中实现了近100%的CCR5淘汰,目标外活动最小.
结论:
- 在那些被测试的人中,egRVLP代表了对CRISPR/Cas传递最有效的复原病毒样颗粒.
- 已建立的表征方法为可预测和更安全的应用程序提供了VLP验证的便利.
- 这项研究推动了用于临床基因编辑策略的VLP的开发.
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