PO.IM01.06 · 免疫学
结合环状RNA与基于硅膜的细胞工程方法制造具有膜结合IL-2和IL-12的CAR-T细胞
Combining circular RNA and a silicon membrane-based cell engineering approach to create CAR-Ts with membrane bound IL-2 and IL-12
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
细胞递送对于细胞疗法和药物开发至关重要，我们开发了一种新型机械穿孔技术，能够将多种货物高效递送至原代细胞和细胞系。该技术使用硅膜暂时使细胞变形，形成孔隙以实现货物向细胞内扩散。重要的是，该方法最大限度地减少了对正常基因表达的干扰，并维持了细胞完整性。它与多种细胞类型兼容，已成功应用于细胞疗法和药物发现。在Portal，我们已成功将CRISPR/Cas9 RNP、mRNA、环状RNA和siRNA递送至T细胞、B细胞、NK细胞和单核细胞。例如，我们在同时递送mRNA和CRISPR-RNP后，在初始T细胞中实现了超过85%的GFP表达和B2M缺失。此外，我们通过递送两种circRNA，证明了CD19 CAR和膜结合IL-2的功能性表达。来自iPSC和HSC的早期数据显示出干细胞工程和分化方面的巨大潜力。使用临床规模原型机，我们实现了每分钟超过1x10⁹个T细胞的递送，在T细胞扩增7天后获得超过50%的敲除效率和90%的GFP表达。我们的平台还通过实现原本无法穿透细胞的货物(如小分子、多肽、PROTAC、DEL、抗体和探针)的递送，解决了药物开发的一大障碍。我们已成功以超过80%的效率递送多种货物，同时维持高细胞活力。该技术已在多种细胞类型中测试，包括癌细胞系、人类免疫细胞和干细胞。机械递送方法的简便性有利于可扩展性以及与液体处理器集成以用于高通量应用。这使得能够对先前不兼容活细胞检测的分子进行快速、自动化筛选，提高药物发现效率。总体而言，Portal的技术提供了一种可扩展、经济高效的解决方案，有望显著推进细胞疗法和药物发现。通过减少制造时间和成本，它可无缝集成到现有的临床和工业设备中，支持下一代细胞疗法的开发，可能彻底改变临床需求高度未满足疾病的治疗。
查看英文原文 English abstract
Cell delivery is crucial for cell therapy and drug development, and we have developed a novel mechanoporation technology that allows efficient delivery of a wide range of cargoes to primary cells and cell lines. This technology uses a silicon membrane to temporarily deform cells, creating pores that enable cargo diffusion into the cells. Importantly, this method minimizes disruption to normal gene expression and maintains cellular integrity. It is compatible with various cell types and has been successfully applied in both cell therapy and drug discovery. At Portal, we have successfully delivered CRISPR/Cas9 RNPs, mRNAs, circular RNAs, and siRNAs into T cells, B cells, NK cells, and monocytes. For instance, we achieved over 85% GFP expression and B2M deletion in naive T cells after simultaneous delivery of mRNA and CRIRPR-RNPs. Additionally, we demonstrated the functional expression of a CD19 CAR and membrane-bound IL-2 by delivering two circRNAs. Early data from iPSCs and HSCs show significant potential for stem cell engineering and differentiation. Using a clinical-scale prototype, we have achieved delivery of over 1x10⁹ T cells per minute, resulting in more than 50% knockout efficiency and 90% GFP expression after 7 days of T cell expansion.Our platform also addresses a major barrier to drug development by enabling the delivery of cargoes that are otherwise impermeable to cells, such as small molecules, peptides, PROTACs, DELs, antibodies, and probes. We have successfully delivered a variety of cargos with over 80% efficiency while maintaining high cell viability. The technology has been tested in several cell types, including cancer cell lines, human immune cells, and stem cells. The simplicity of the mechanical delivery method facilitates scalability and integration with liquid handlers for high-throughput applications. This enables rapid and automated screening of molecules previously incompatible with live cell assays, improving drug discovery efficiency. Overall, Portal's technology offers a scalable, cost-effective solution with the potential to significantly advance both cell therapy and drug discovery. By reducing manufacturing time and cost, it seamlessly integrates into existing clinical and industrial equipment, supporting the development of next-generation cell-based treatments that could revolutionize the treatment of diseases with high unmet clinical needs.
利益披露 Disclosure
A. Sharei, None.