Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Impact of Formulation Conditions on Lipid Nanoparticle Characteristics and Functional Delivery of CRISPR RNP for Gene Knock-Out and Correction

Version 1 : Received: 10 December 2021 / Approved: 14 December 2021 / Online: 14 December 2021 (18:24:56 CET)

A peer-reviewed article of this Preprint also exists.

Walther, J.; Wilbie, D.; Tissingh, V.S.J.; Öktem, M.; van der Veen, H.; Lou, B.; Mastrobattista, E. Impact of Formulation Conditions on Lipid Nanoparticle Characteristics and Functional Delivery of CRISPR RNP for Gene Knock-Out and Correction. Pharmaceutics 2022, 14, 213. Walther, J.; Wilbie, D.; Tissingh, V.S.J.; Öktem, M.; van der Veen, H.; Lou, B.; Mastrobattista, E. Impact of Formulation Conditions on Lipid Nanoparticle Characteristics and Functional Delivery of CRISPR RNP for Gene Knock-Out and Correction. Pharmaceutics 2022, 14, 213.

Journal reference: Pharmaceutics 2022, 14, 213
DOI: 10.3390/pharmaceutics14010213

Abstract

The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse ge-netic disorders. However, for gene therapy applications an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. Once there, the ribonucleoprotein complex (RNP) can be transported into the nucleus. Lipid nanoparticles (LNP) serve as promising candidates for delivery of CRISPR-Cas9 RNP. These delivery vehicles have been optimized for the delivery of nucleic acids, such as mRNA. Co-delivery of Cas9 encoding mRNA with the accompanying sgRNA leads to translation of the Cas9 protein and formation of the Cas9 RNP inside the cell. Only recently, direct delivery of the CRISPR-Cas9 RNP complexes has been explored, which requires adjustments to the LNP formulation. In this study, the importance of buffer composition and cationic charge during RNP and ssDNA en-trapment in LNP are demonstrated. After optimizing several formulation parameters, LNP were prepared that were colloidally stable in human plasma and efficiently deliver the SpCas9 RNP and ssDNA for HDR-correction in reporter cells. Under optimal formulation conditions, gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively were achieved at nanomolar CRISPR-Cas9 RNP concentrations.

Keywords

CRISPR; LNP; formulation; NHEJ; HDR; AF4; delivery

Subject

ENGINEERING, Biomedical & Chemical Engineering

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