Enhancing RNA-lipid nanoparticle delivery: Organ- and cell-specificity and barcoding strategies

Abstract

Recent advancements in RNA therapeutics highlight the critical need for precision gene delivery systems that target specific organs and cells. Lipid nanoparticles (LNPs) have emerged as key vectors in delivering mRNA and siRNA, offering protection against enzymatic degradation, enabling targeted delivery and cellular uptake, and facilitating RNA cargo release into the cytosol. This review discusses the development and optimization of organ- and cell-specific LNPs, focusing on their design, mechanisms of action, and therapeutic applications. We explore innovations such as DNA/RNA barcoding, which facilitates high-throughput screening and precise adjustments in formulations. We address major challenges, including improving endosomal escape, minimizing off-target effects, and enhancing delivery efficiencies. Notable clinical trials and recent FDA approvals illustrate the practical applications and future potential of LNP-based RNA therapies. Our findings suggest that while considerable progress has been made, continued research is essential to resolve existing limitations and bridge the gap between preclinical and clinical evaluation of the safety and efficacy of RNA therapeutics. This review highlights the dynamic progress in LNP research. It outlines a roadmap for future advancements in RNA-based precision medicine.

Keywords: DNA barcoding; Gene delivery; Lipid carriers; Personalized medicine; RNA therapies; Site-specific.

Fig. 1.. Mechanism of Action of mRNA and siRNA therapeutics.

mRNA and siRNA therapeutics both aim to alter protein functions by enabling protein translation or inhibiting protein translation. When formulated in lipid nanoparticles, both enter cells via endocytosis, which makes the “endosomal escape” of RNA-LNPs the primary biological barrier in their bioavailability. By carefully designing RNA sequences, mRNA serves as multifunctional platforms to produce intracellular proteins or antigens, membrane-bounded proteins or receptors, and secreted proteins that could also be recycled and endocytosed by the cell (left panel). On the contrary, siRNA enabled sequence-specific degradation of mRNA through the recruitment of RNA-induced Silencing Complex (RISC), leading to down-regulation of protein expression (right panel), which could serve as a stand-alone therapy or as emergency brakes for mRNA therapies to counteract any unintended consequences of mRNA therapy by silencing the same mRNA (This figure is created with BioRender.com).

Fig. 2.. A 60-year journey from liposomes to ionizable lipid nanoparticles for RNA delivery.

This retrospective timeline summarizes the development of liposomes and lipid nanoparticle formulation, with selected milestones highlighting key discoveries for drug and gene delivery. Full itemized information is summarized in Table S1.

Fig. 3.. Demonstration of the Critical Packing Parameter (CPP) and its application in designing ionizable lipids for effective mRNA delivery in lipid nanoparticles.

CPP is a determinant of lipid molecular arrangement in aqueous environments, defined by the equation N_s = V_c / a_e * L_c, where V_c = the volume of the hydrophobic chain, L_c = length of the hydrophobic chain, a_e = the effective surface area of the hydrophilic head. The most likely self-assembled structures of lipid molecules in water may differ depending on their corresponding CPP. (a) Gene-delivering ionizable lipids such as MC-3 and SM-102 have a much larger V_c and a much smaller head group, yielding a CPP >1, conducive to forming pH-responsive, non-lamellar structures like inverse micelles, with internal ionizable heads and external hydrophobic tails. [78,268]. (b) In contrast, membrane lipids such as DSPC with a CPP approximately equal to 1 result in a bilayer structure due to its cylindrical shape, with hydrophilic heads at the water interface and hydrophobic tails tucked away. (c) At lower pH levels, the ionizable amines on the SM-102 headgroups become cationic, facilitating electrostatic complexation with anionic nucleic acids. This interaction ensures the nucleic acids are securely encapsulated within the interior, with the hydrophobic tails of SM-102 oriented outward. However, such a hydrophobic exterior is inherently unstable in aqueous environments. To overcome this, additional membrane-forming lipids are incorporated into the nanoparticle formulation. These lipids assemble into a stabilizing hydrophilic monolayer around the exterior, providing a compatible interface with the aqueous medium for enhanced colloidal stabilities, which is essential for the practical application of the lipid nanoparticles for mRNA delivery.

Fig. 4.

Lipids for formulating RNA delivering ionizable lipid nanoparticles.

Fig. 5.. Biodistribution tracers for developing organ-specific and cell-specific LNPs.

a. Spatial resolution of commonly used molecular tracers for PK/PD studies. b. Molecular labeling techniques for tracking lipids in the LNPs. c. Comparing bioluminescent-based and fluorescent-based imaging techniques for tracking RNA delivery by LNPs. d. Illustration of using Cre-Lox system for detecting mRNA delivery. e. The DNA-barcoding workflow for HTS LNP formulation screening in vivo. (panels and d are partially created with BioRender.com).

Fig. 6.. A six-decade-long journey from discovery to clinical approvals of mRNA and siRNA therapeutics.

This retrospective timeline summarizes the development of mRNA and siRNA therapeutics, with selected milestones highlighting key developments for enabling their clinical applications. Full itemized information is summarized in Table S2.

Fig. 7.. Clinical Landscape of mRNA and siRNA Therapeutics.

A. Search terms for clinicaltrial.gov using keywords included “siRNA,” “mRNA,” “RNAi,” “messenger RNA,” and “interfering RNA.”. Exclusions were made for trials that did not involve mRNA or siRNA as therapeutic— remaining trials were included for further analysis (detailed in Fig. S1). B. mRNA therapies, C. mRNA/cell therapies, and D. siRNA therapies. E. Analyzing indications in mRNA therapy, the top 3 indications were classified by phase and marked in F. Conducting a similar analysis for mRNA/cell therapy and siRNA therapy, with mRNA/cell therapy labeled in G. H and siRNA labeled in I.J. Inclusion of trials: up to April 24, 2024. Detailed classification is available via the online supporting information.