Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany.
Department of Neurosurgery, University Medical Centre Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany.
Int J Mol Sci. 2023 Jul 13;24(14):11399. doi: 10.3390/ijms241411399.
The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and "enhanced" egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application.
CRISPR/Cas 系统在医学领域具有广泛的应用前景，但由于缺乏安全有效的载体系统来介导其成分的短期表达，其临床转化受到了阻碍。最近，不同的病毒样颗粒（VLPs）已被引入作为递送 CRISPR/Cas 基因组编辑组件的有前途的载体。在这里，我们对三种不同类型的基于逆转录病毒（R）的 VLPs 进行了表征和直接比较，其中两种来源于γ-逆转录病毒鼠白血病病毒（gRVLPs 和“增强”egRVLPs），一种来源于慢病毒人类免疫缺陷病毒（HIV）（LVLPs）。首先，我们统一并优化了不同 RVLPs 的生产。为了确保生产的 RVLPs 具有最大的可比性，我们采用了多种分析方法，包括纳米颗粒跟踪分析（NTA）、多参数成像流式细胞术（IFC）和 Cas9-ELISA，以分析它们的形态、表面组成、大小和浓度。接下来，我们在 293T 模型细胞中比较测试了三种靶向不同基因的 RVLPs。使用相同的 gRNA，我们发现 egRVLPs 介导的编辑效率最高。功能分析表明，更好的货物（即 Cas9）转移和/或释放是其性能优越的原因。最后，我们利用临床上相关的 C-C 基序趋化因子受体 5（CCR5）作为靶点，在人诱导多能干细胞（hiPSC）中比较了三种 RVLPs 的靶上和靶外活性。同样，egRVLPs 促进了最高的、几乎 100%的敲除率，重要的是，靶外活性最小。总之，在直接比较中，egRVLPs 是最有效的 RVLPs。此外，我们建立了深入表征 VLP 的方法，促进了它们的验证，从而使它们的应用更具可预测性和安全性。
