In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels

Tanja Rothgangl ,S Egli ,Martina Hruzova ,Norbert Pardi ,Andreas E Moor ,Beat Thöny ,Drew Weissman ,Martin Pačesa ,Nina Frey ,Paulo J.c Lin ,Susanne Kreutzer ,Kevin Holden ,Markus Stoffel ,Noëlle Bakker ,Anastasia P Kadina ,Sean C Semple ,Melissa K Dennis ,Costanza Borrelli ,Lucas Kissling ,Ying K Tam ,Weihong Qi ,Lukas Villiger ,Zacharias Kontarakis ,Dominik Witzigmann ,Martin Jínek ,Johannes Häberle ,Rurika Oka ,Denis V Victorov ,Ruben Van Boxtel ,Daniela Lenggenhager ,John Walker ,Gerald Schwank

doi:10.1038/s41587-021-00933-4

Abstract

Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

Highlights

Programmable CRISPR–Cas nucleases enable genome editing by generating double-stranded DNA breaks at the target locus[1]
For the human Pcsk[9] splice donor site of intron 1, we observed similar editing efficiencies with the corresponding single guide RNA (sgRNA) in human HepG2 cells (89 ± 1.6%; Supplementary Fig. 2f–h), and, because the target sequence is perfectly conserved to non-human primates (NHPs) (Supplementary Fig. 2b), we selected sgRNA_mP01 and sgRNA_hP01 for our in vivo experiments in mice and cynomolgus macaques, respectively (Fig. 1a)
Six weeks after associated virus (AAV)-based adenine base editors (ABEs) delivery, genomic DNA was isolated from the liver, and the target locus was analyzed by targeted amplicon deep sequencing

Summary

Introduction

Programmable CRISPR–Cas nucleases enable genome editing by generating double-stranded DNA breaks at the target locus[1]. Considering that most pathogenic point mutations are CG to TA conversions, ABEs are of particular interest for in vivo genome editing therapies[8]. For clinical application of base editing, the potential generation of off-target mutations represents a major concern. Off-target mutations could be single guide RNA (sgRNA) dependent[11,12] or sgRNA independent[13,14,15] and are influenced by the levels and duration of base editor expression[16,17,18]. The risks of generating off-target mutations in therapies are likely to depend on the delivery method and dose. We report that lipid nanoparticle (LNP)-mediated delivery of ABE-encoding nucleoside-modified mRNA, together with a chemically stabilized sgRNA, enables efficient editing in mice and NHPs without inducing off-target mutations on genomic DNA

Methods

Results

Conclusion