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RGD Peptide-Based Lipids For Targeted mRNA Delivery and Gene Editing Applications

A combinatorial library of RGD peptide (arginine-glycine-aspartic acid)-based lipids designed to formulate lipid nanoparticles for targeted mRNA delivery to the liver and the spleen.

Problem:

Lipid Nanoparticles (LNPs) are becoming an established choice of vehicle to deliver mRNA-based therapeutics, including vaccines, protein replacement therapies, and gene editing therapies. An outstanding challenge remains to deliver the LNP and its mRNA cargo in a site-specific manner. Non-targeted delivery can compromise transfection and genome editing efficiency and result in off-target toxicity.

Solution:

Mitchell et al., developed a library of RGD-based LNPs whereby the RGD motif serves as a targeting ligand to direct the LNP to cells with cell-surface integrins, opening the potential for cancer cell targeting. The inventors have identified a lead candidate that demonstrates an improved safety profile and transfection efficiency compared to a non-targeted LNP.

Technology:

The technology comprises a library of novel RGD-based lipids and a method of synthesizing RGD-based LNPs. The assembly process involves addition of the RGD lipid to the conventional four lipid components and subsequent microfluidic mixing with the mRNA cargo. In vitro evaluation of a lead 1A RGD LNP showed significantly improved cell viability, mRNA delivery, and gene editing efficiency compared to a non-targeted LNP. The lead RGD LNP also resulted in greater whole-body expression in vivo in mice, with selectivity toward the liver and the spleen.

Advantages:

Efficient mRNA encapsulation (close to 90%)
Improved transfection and gene editing efficiencies compared to a non-targeted LNP
Up to 90% gene editing efficiency demonstrated in vitro
Targeted delivery to the liver and the spleen
Improved cell viability
Potential application in cancer cell targeting

(A) Schematic of the assembly process for generating RGD-based LNPs loaded with an mRNA cargo. (B) RGD-based LNPs target cells with cell-surface integrin, allowing for potential tumor targeting. (C-D) The lead 1A RGD LNP demonstrates: (C) Improved transfection efficiency in vitro; and, (D) Increased whole body expression in mice in vivo compared to a non-targeted LNP control. (E) Delivery of 1A RGD LNP predominates in the mouse liver and spleen. (F) Toward gene editing applications, the RGD LNP shows effective co-delivery of Cas9 mRNA and single guide RNA, achieving up to 90% knockout efficiency in vitro and outperforming a non-targeted LNP control.