Jia Jinlin, Hao Yanzhe, Zhang Lu, Cao Xiaofang, An Lisha, Wang Hu, Ma Qi, Jin Xiaohua, Ma Xu
National Research Institute for Family Planning, Beijing, 100081, China.
Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, 100005, China.
Eur J Med Res. 2025 Apr 4;30(1):242. doi: 10.1186/s40001-025-02499-2.
The development of gene editing tools such as CRISPR-Cas9 and base editors (BE) is critical for genetic diseases and cancer. Lentivirus-like particles (LVLPs) grows into an auspicious platform for delivering mRNA or ribonucleic proteins (RNPs) due to it integrates the advantage of viral and non-viral vectors. Current LVLP systems predominantly utilize HIV-Gag and Pol proteins. However, the reverse transcriptase and integrase of Pol, pose risks of genomic integration and potential tumorigenesis. Enhancing the safety of VLP system is essential. This study focuses on improving the LVLP to minimize these risks.
We implemented a Gag-Only strategy, constructing LVLPs with HIV-Gag protein, thereby eliminating the integration risks linked to Pol. By leveraging the interactions between MS2-MCP (MS2 coat protein), PP7 and PP7 BP (PP7 binding protein), and the psi (HIV packaging signal) with HIV-Gag, we encapsulated PAMless andesine base editor (CE-8e-SpRY) mRNA and sgRNA targeting the PD1 start codon (ATG) into the LVLP. Using recombinant lentiviral vector technology, we developed a stable PD1-expressing 293T cell line (PD1-293T) to assess the editing efficiency of LVLP.
The psi-LVLP demonstrated effective packaging capabilities, achieving 15% base editing efficiency in 293T cells. By optimizing plasmid ratios, we observed increased CE-8e-SpRY mRNA copy numbers, with 30% base editing efficiency. Additionally, the integration of HDVrz (hepatitis delta virus ribozyme) and psi into sgRNA (HDVrz-psi-LVLP) substantially enhanced sgRNA copy numbers, resulting in approximately 50% base editing efficiency in 293T cells and 20% base editing efficiency in Jurkat cells. Mendelian randomization analyses revealed significant genetic correlations between PD1, B2M, CIITA, and TIGIT genes with various cancer risks. Furthermore, HDVrz-psi-LVLPs targeting the start codons of B2M, CIITA, and TIGIT exhibited high base editing activity in both Jurkat and 293T cells.
In conclusion, this optimized platform effectively encapsulates CE-8e-SpRY mRNA and sgRNA, achieving high editing efficiencies across multiple genes and cell types. We introduce a safer and more efficient gene editing tool delivery system by constructing LVLPs based on the Gag-Only strategy. Our study presents a promising implication for cancer immunotherapy.
CRISPR-Cas9和碱基编辑器（BE）等基因编辑工具的发展对治疗遗传疾病和癌症至关重要。慢病毒样颗粒（LVLP）由于整合了病毒载体和非病毒载体的优势，正成为一种用于递送信使核糖核酸（mRNA）或核糖核蛋白（RNP）的理想平台。目前的LVLP系统主要利用HIV- Gag和Pol蛋白。然而，Pol的逆转录酶和整合酶存在基因组整合风险和潜在的肿瘤发生风险。提高病毒样颗粒（VLP）系统的安全性至关重要。本研究聚焦于改进LVLP以将这些风险降至最低。
我们实施了仅Gag策略，构建含有HIV-Gag蛋白的LVLP，从而消除与Pol相关的整合风险。通过利用MS2-MCP（MS2外壳蛋白）、PP7和PP7 BP（PP7结合蛋白）之间的相互作用，以及psi（HIV包装信号）与HIV-Gag之间的相互作用，我们将无PAM的安第斯碱基编辑器（CE-8e-SpRY）mRNA和靶向PD1起始密码子（ATG）的sgRNA封装到LVLP中。利用重组慢病毒载体技术，我们构建了一个稳定表达PD1的293T细胞系（PD1-293T）来评估LVLP的编辑效率。
psi-LVLP表现出有效的包装能力，在293T细胞中实现了15%的碱基编辑效率。通过优化质粒比例，我们观察到CE-8e-SpRY mRNA拷贝数增加，碱基编辑效率达到30%。此外，将丁型肝炎病毒核酶（HDVrz）和psi整合到sgRNA中（HDVrz-psi-LVLP）显著提高了sgRNA拷贝数，在293T细胞中碱基编辑效率约为50%，在Jurkat细胞中为20%。孟德尔随机化分析显示，PD1、B2M、CIITA和TIGIT基因与各种癌症风险之间存在显著的遗传相关性。此外，靶向B2M、CIITA和TIGIT起始密码子的HDVrz-psi-LVLP在Jurkat细胞和293T细胞中均表现出高碱基编辑活性。
总之，这个优化平台有效地封装了CE-8e-SpRY mRNA和sgRNA，在多个基因和多种细胞类型中实现了高编辑效率。我们通过基于仅Gag策略构建LVLP，引入了一种更安全、更高效的基因编辑工具递送系统。我们的研究为癌症免疫治疗提供了有前景的启示。
