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

Johanna Walther,Heleen Van Der Veen,Bo Lou,Vincent S J Tissingh,Mert Öktem,Enrico Mastrobattista,Danny Wilbie

doi:10.3390/pharmaceutics14010213

Abstract

The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse genetic 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. In this study, we optimized the formulation conditions of lipid nanoparticles (LNP) for delivery of ready-made CRISPR-Cas9 ribonucleic protein (RNP). The buffer composition during complexation and relative DOTAP concentrations were varied for LNP encapsulating in-house produced Cas9 RNP alone or Cas9 RNP with additional template DNA for gene correction. The LNP were characterized for size, surface charge, and plasma interaction through asymmetric flow field flow fractionation (AF4). Particles were functionally screened on fluorescent reporter cell lines for gene knock-out and gene correction. This revealed incompatibility of RNP with citrate buffer and PBS. We demonstrated that LNP for gene knock-out did not necessarily require DOTAP, while LNP for gene correction were only active with a low concentration of DOTAP. The AF4 studies additionally revealed that LNP interact with plasma, however, remain stable, whereby HDR template seems to favor stability of LNP. Under optimal formulation conditions, we achieved gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively, at nanomolar concentrations of the CRISPR-Cas9 RNP.

Highlights

The clustered regularly interspaced short palindromic repeats (CRISPR) associated (Cas) endonuclease proteins, such as Cas9, have emerged in recent years as a viable therapeutic option for genetic diseases
To study the long-term stability of in-house produced SpCas9, SpCas9, purified SpCas9 from a representative batch was snap frozen in liquid nitrogen purified SpCas9 from a representative batch was snap frozen in liquid nitrogen and stored and stored in aliquots at −80 °C until needed for analysis of protein size, activity, and for in aliquots at −80 ◦ C until needed for analysis of protein size, activity, and for use in lipid nanoparticles (LNP)
RNP mixed at different NaCl concentrations did not lose activity (Supplementary Figure S6B). Taken together these findings indicate that pH or ionic strength alone do not account for the loss of Cas9 activity in the formulations

Summary

Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR) associated (Cas) endonuclease proteins, such as Cas, have emerged in recent years as a viable therapeutic option for genetic diseases. The Cas endonuclease was first identified as a bacterial defense mechanism against viral infections and has been repurposed into a powerful tool to cleave DNA in an RNA-guided fashion in various cell types. The Cas protein, together with a guide RNA molecule, forms an active ribonucleoprotein (RNP). DNA cleavage is mediated by recognition of a 20-nucleotide sequence between the guide RNA and the host DNA, which hybridizes and allow the nuclease to attach to its DNA target. The presence of a protospacer-adjacent motif in the host DNA is necessary to facilitate the conformational change in the nuclease to introduce a double strand break in its target [2]. When the genomic DNA is cleaved by the Cas enzyme, the host DNA-damage repair response is activated [3]. The most prominent pathways are the canonical non-homologous end-joining (c-NHEJ) pathway, the microhomology-mediated end joining (MMEJ) pathway, and homology-directed repair

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Results

Discussion

Conclusion