Lipo-Xenopeptide Polyplexes for CRISPR/Cas9 based Gene editing at ultra-low dose
Janin Germer, Anna-Lina Lessl, Jana Pöhmerer, Melina Grau, Eric Weidinger, Miriam Höhn, Mina Yazdi, Martino Alfredo Cappelluti, Angelo Lombardo, Ulrich Lächelt, Ernst Wagner
Abstract
Double pH-responsive xenopeptide carriers containing succinoyl tetraethylene pentamine (Stp) and lipo amino fatty acids (LAFs) were evaluated for CRISPR/Cas9 based genome editing. Different carrier topologies, variation of LAF/Stp ratios and LAF types as Cas9 mRNA/sgRNA polyplexes were screened in three different reporter cell lines using three different genomic targets ( Pcsk9 , eGFP, mdx exon 23). One U-shaped and three bundle (B2)-shaped lipo-xenopeptides exhibiting remarkable efficiencies were identified. Genome editing potency of top carriers were observed at sub-nanomolar EC 50 concentrations of 0.4 nM sgRNA and 0.1 nM sgRNA for the top U-shape and top B2 carriers, respectively, even after incubation in full (≥ 90%) serum. Polyplexes co-delivering Cas9 mRNA/sgRNA with a single stranded DNA template for homology directed gene editing resulted in up to 38% conversion of eGFP to BFP in reporter cells. Top carriers were formulated as polyplexes or lipid nanoparticles (LNPs) for subsequent in vivo administration. Formulations displayed long-term physicochemical and functional stability upon storage at 4 °C. Importantly, intravenous administration of polyplexes or LNPs mediated in vivo editing of the dystrophin gene, triggering mRNA exon 23 splicing modulation in dystrophin-expressing cardiac muscle, skeletal muscle and brain tissue. • pH-responsive xenopeptides for Cas9 mRNA/sgRNA formulation as polyplex or LNP. • genome editing of three different gene targets in different cell lines. • highly active in full serum, potency at amazingly low (100 pM) concentrations. • intravenous or intramuscular application for editing dystrophin exon 23 splice site. • genome modification and exon skipping in cardiac muscle, skeletal muscle and brain.