High-Throughput Screening of Lipid Nanoparticles for Efficient CRISPR RNA Delivery In Vitro and in Vivo

Category: Nonviral Delivery: E1 - Lipid nanoparticles (including Engineering, therapeutic delivery, Manufacturing, CMC considerations)

Authors: Balgees Khader, Wei Nei, Baisong Lu

Affiliation: Wake Forest Institute for Regenerative Medicine.

Keywords: CRISPR/Cas9; Gene Correction/Modification/Targeting; Lipid Nanoparticles (LNP)

Abstract Body: 

Introduction:

Efficient and precise delivery of CRISPR/Cas9 components remains one of the critical challenges toward the advancement of gene-editing therapies. Here, we have developed a high-throughput Barcode-Integration Nanoparticle Screen (BINS) method to evaluate a library of 96 lipid nanoparticles (LNPs) for their delivery efficiency across in vitro and in vivo models.

Methods:

For proof of concept, we designed a library of 96 LNPs made from 24 distinct ionizable lipids, without and with one of three different SORT (Selective Organ Targeting) lipids: cationic-SORT, anionic-SORT, and brain-targeting SORT. Each LNP encapsulated CRISPR/Cas9 mRNA, sgRNA, and a unique double-stranded oligonucleotide barcode. The pooled LNPs were delivered to retinal pigment epithelial (RPE) Cells and human skeletal muscle precursor cells (SkMPCs), and mice through  in vivo  systemic intravenous injections. Integrated barcode DNA was analyzed via PCR and Next-Generation Sequencing (NGS) to quantify integrated barcode, which reflects the delivery efficiency of the barcoded LNP.

Results:

We observed different barcode profiles integration in different cells and tissues. Negative control LNPs without sgRNA showed no barcode integration in all cells/organs detected. Whereas many LNPs showed low or no barcode integration in all cells/tissues analyzed, LNPs with high barcode integration frequencies were found for RPEs, SkMPCs and different organs. These LNPs will be validated for their delivery efficiency to their respective target cells.

Conclusions:

This highlights work the potential of the BINS method in screening for candidate LNPS for targeted RNA delivery. The screening of multiple ionizable lipids and SORT lipids together with barcoding-based tracking offers an opportunity to optimize LNP formulations for specific organ systems. The LNPs found this way will have translational values ​​in precision medicine.