Qin Jingya, Xue Lulu, Gong Ningqiang, Zhang Hanwen, Shepherd Sarah J, Haley Rebecca M, Swingle Kelsey L, Mitchell Michael J
Department of Bioengineering, University of Pennsylvania Philadelphia PA 19104 USA
Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania Philadelphia Pennsylvania 19104 USA.
RSC Adv. 2022 Sep 7;12(39):25397-25404. doi: 10.1039/d2ra02771b. eCollection 2022 Sep 5.
mRNA therapeutics are promising platforms for protein replacement therapies and gene editing technologies. When delivered non-viral gene delivery systems, such as lipid nanoparticles (LNPs), mRNA therapeutics are easy to produce and show low toxicity and immunogenicity. However, LNPs show limited delivery efficiency and tissue specificity in certain applications. To overcome this, we designed RGD peptide (Arg-Gly-Asp) based ionizable lipids, which can be formulated into LNPs for integrin binding on cells and targeted mRNA delivery. RGD-LNPs were formulated using microfluidic devices and screened for size, mRNA encapsulation efficiency, transfection efficiency, and cell viability. A lead candidate, 1A RGD-based hybrid LNP, showed effective mRNA encapsulation and transfection, and was selected for further testing, including the co-delivery of Cas9 mRNA and sgRNA for gene editing applications. , 1A RGD-based hybrid LNP outperformed a non-targeted control LNP and showed GFP knockout efficiencies up to 90%. Further, the improved cellular uptake was reversed in the presence of soluble RGD, supporting the hypothesis that this improved uptake is RGD-dependent. , 1A RGD-based hybrid LNPs showed comparable mRNA delivery to the liver and spleen, when compared to a non-targeted control, and had increased expression in the whole body. Overall, this RGD-based hybrid LNP system is a promising platform for targeted mRNA delivery, which may allow for mRNA-based protein replacement and gene editing in a more efficient and specific manner with reduced off-target effects.
信使核糖核酸（mRNA）疗法是蛋白质替代疗法和基因编辑技术中很有前景的平台。当通过非病毒基因递送系统（如脂质纳米颗粒（LNPs））递送时，mRNA疗法易于生产，且毒性和免疫原性较低。然而，在某些应用中，LNPs的递送效率和组织特异性有限。为克服这一问题，我们设计了基于RGD肽（精氨酸-甘氨酸-天冬氨酸）的可电离脂质，其可被制成LNPs用于细胞上的整合素结合和靶向mRNA递送。使用微流控装置制备了RGD-LNPs，并对其尺寸、mRNA包封效率、转染效率和细胞活力进行了筛选。一种领先的候选物，即基于1A RGD的混合LNP，显示出有效的mRNA包封和转染，并被选用于进一步测试，包括用于基因编辑应用的Cas9 mRNA和sgRNA的共递送。基于1A RGD的混合LNP优于非靶向对照LNP，显示出高达90%的绿色荧光蛋白（GFP）敲除效率。此外，在可溶性RGD存在的情况下，改善的细胞摄取被逆转，支持了这种改善的摄取是RGD依赖性的假设。与非靶向对照相比，基于1A RGD的混合LNP在肝脏和脾脏中的mRNA递送相当，且在全身的表达增加。总体而言，这种基于RGD的混合LNP系统是一种有前景的靶向mRNA递送平台，其可能允许以更高效、更特异的方式进行基于mRNA的蛋白质替代和基因编辑，同时减少脱靶效应。
