Optimization of lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes for in vivo delivery of a CRISPR/Cas9 system targeting hepatitis B virus

TL;DR

Researchers optimized lipid nanoparticles to deliver a CRISPR/Cas9 system targeting hepatitis B virus in mice, achieving significant suppression of viral replication.

Contribution

A new lipid nanoparticle formulation (CL4F11_ζ−2) combined with heat-treated RNA significantly improved in vivo CRISPR/Cas9 delivery for HBV suppression.

Findings

CL4F11_ζ−2 LNP/WJ11-Cas9 significantly reduced serum HBV levels in mice.

Heat treatment of WJ11 sgRNA enhanced the efficacy of LNP-based CRISPR/Cas9 delivery.

The optimized LNP formulation reduced hepatic HBV DNA, cccDNA, HBsAg, and HBcrAg levels.

Abstract

•LNP-mediated in vivo delivery of gRNAs/Cas9 systems has been investigated.•We investigated 3 LNP candidates for inhibition of HBV replication in vivo.•CL4F11_ζ−2 LNP/WJ11-Cas9 showed significant suppression of serum HBV level.•Heat treatment of WJ11 sgRNA may improve the efficacy of LNP-Cas9.

LNP-mediated in vivo delivery of gRNAs/Cas9 systems has been investigated.

We investigated 3 LNP candidates for inhibition of HBV replication in vivo.

CL4F11_ζ−2 LNP/WJ11-Cas9 showed significant suppression of serum HBV level.

Heat treatment of WJ11 sgRNA may improve the efficacy of LNP-Cas9.

Patients with chronic hepatitis B virus (HBV) infection may benefit from clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene therapy. We previously identified a guide RNA (WJ11) that suppressed HBV replication in vitro and in vivo; however, we were unable to achieve delivery at clinically feasible doses in vivo using an adeno-associated virus (AAV) vector. Lipid nanoparticle (LNP)-based WJ11/Cas9 ribonucleoprotein-oligonucleotide complex delivery suppressed HBV replication by 2–3-fold more than did AAV-based delivery. In the present study, we investigated the HBV replication-suppressive effects of LNP/WJ11/Cas9 complexes after intravenous administration to persistently HBV genotype C-infected humanized chimeric mice. CL4H6 (ionizable lipid) LNPs were selected as the first candidate for WJ11/Cas9 delivery based on their reported high encapsulation efficiency; however, no significant anti-HBV effect was noted in serum or hepatic tissue. The ionizable lipid candidate CL4F11_ε-3 improved absolute serum HBV values to a certain degree but had no significant effect on hepatic HBV DNA or covalently closed circular (ccc)DNA levels. CL4F11_ζ-2 LNP/WJ11/Cas9, a new complex prepared through structural optimization of the ionizable lipid and heat treatment of WJ11, showed suppressive effect for serum viral load along with a reduction of hepatic HBV DNA, HBV cccDNA, HBsAg, and HBcrAg levels when compared with controls. Therefore, LNP-based delivery of this CRISPR/Cas9 formula holds promise for the treatment of chronic HBV infection.

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