A Combinatorial Library of Lipid Nanoparticles for RNA Delivery to Leukocytes

Abstract

Lipid nanoparticles (LNPs) are the most advanced nonviral platforms for small interfering RNA (siRNA) delivery that are clinically approved. These LNPs, based on ionizable lipids, are found in the liver and are now gaining much attention in the field of RNA therapeutics. The previous generation of ionizable lipids varies in linker moieties, which greatly influences in vivo gene silencing efficiency. Here novel ionizable amino lipids based on the linker moieties such as hydrazine, hydroxylamine, and ethanolamine are designed and synthesized. These lipids are formulated into LNPs and screened for their efficiency to deliver siRNAs into leukocytes, which are among the hardest to transfect cell types. Two potent lipids based on their in vitro gene silencing efficiencies are also identified. These lipids are further evaluated for their biodistribution profile, efficient gene silencing, liver toxicity, and potential immune activation in mice. A robust gene silencing is also found in primary lymphocytes when one of these lipids is formulated into LNPs with a pan leukocyte selective targeting agent (β7 integrin). Taken together, these lipids have the potential to open new avenues in delivering RNAs into leukocytes.

CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy

“…To overcome the cargo limitation of currently available LNP formulations, LNPs were designed to coencapsulate Cas9 mRNA and sgRNA, using ionizable cationic lipids from a novel ionizable amino lipid library ( Fig. 1A) (12). This library was constructed using a novel class of ionizable amino lipids based on hydrazine, hydroxylamine, and ethanolamine linkers with a linoleic fatty acid chain and amine head groups (12).…”

“…1A) (12). This library was constructed using a novel class of ionizable amino lipids based on hydrazine, hydroxylamine, and ethanolamine linkers with a linoleic fatty acid chain and amine head groups (12). Lipids 1, 6, 8, and 10 were the top hits of the screen and were chosen for further evaluation for CRISPR-Cas9 gene editing (Fig.…”

“…DLin-MC3-DMA (MC3) and Lipid 8 were synthesized according to a previously described method (12,23). Cholesterol, DSPC (1,2distearoyl-sn-glycero-3-phosphocholine), polyethylene glycol (PEG)-DMG (1,2-dimyristoyl-rac-glycerol), and DSPE (1,2-distearoyl-snglycero-3-phosphoethanolamine)-PEG were purchased from Avanti Polar Lipids Inc.…”

Design of SARS-CoV-2 RBD mRNA Vaccine Using Novel Ionizable Lipids

“…Our lab previously demonstrated the design and synthesis of novel ionizable amino lipids for efficient siRNA delivery to leukocyte subsets [12] . Herein, we have selected several structurally different ionizable lipids and screened for in vivo mRNA-LNPs delivery for vaccine applications.…”

Efficient Polymer-Mediated Delivery of Ribonucleoprotein Payloads Through Combinatorial Design & Parallelized Experimentation

“…Recognizing the urgent clinical need for non-viral vectors, we deployed parallelized experimentation and machine learning to rapidly discover high-performing polymeric vehicles for ribonucleoproteins (RNPs). We draw attention to previous studies where combinatorial libraries of cationic lipids, [26][27][28][29][30][31] polymers, 23,[32][33][34][35][36][37][38][39][40] and gold nanoparticles, [41][42][43] were screened to identify high-performing synthetic gene delivery vectors rapidly. Although the application of high-throughput approaches is becoming more widespread in gene delivery, 44 the elucidation of structure-function relationships from large datasets and the development of predictive models that identify correlations between biological responses and polymer attributes has received scant attention.…”