Lipo-Xenopeptide Polyplexes for CRISPR/Cas9 based Gene editing at ultra-low dose.

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 EC50 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.

Lipoamino bundle LNPs for efficient mRNA transfection of dendritic cells and macrophages show high spleen selectivity.

Messenger RNA (mRNA) is a powerful tool for nucleic acid-based therapies and vaccination, but efficient and specific delivery to target tissues remains a significant challenge. In this study, we demonstrate lipoamino xenopeptide carriers as components of highly efficient mRNA LNPs. These lipo-xenopeptides are defined as 2D sequences in different 3D topologies (bundles or different U-shapes). The polar artificial amino acid tetraethylene pentamino succinic acid (Stp) and various lipophilic tertiary lipoamino fatty acids (LAFs) act as ionizable amphiphilic units, connected in different ratios via bisamidated lysines as branching units. A series of more lipophilic LAF4-Stp1 carriers with bundle topology is especially well suited for efficient encapsulation of mRNA into LNPs, facilitated cellular uptake and strongly enhanced endosomal escape. These LNPs display improved, faster transfection kinetics compared to standard LNP formulations, with high potency in a variety of tumor cell lines (including N2a neuroblastoma, HepG2 and Huh7 hepatocellular, and HeLa cervical carcinoma cells), J774A.1 macrophages, and DC2.4 dendritic cells. High transfection levels were obtained even in the presence of serum at very low sub-microgram mRNA doses. Upon intravenous application of only 3 µg mRNA per mouse, in vivo mRNA expression is found with a high selectivity for dendritic cells and macrophages, resulting in a particularly high overall preferred expression in the spleen.

Molecular Chameleon Carriers for Nucleic Acid Delivery: The Sweet Spot between Lipoplexes and Polyplexes.

Taking advantage of effective intracellular delivery mechanisms of both cationizable lipids and polymers, highly potent double pH-responsive nucleic acid carriers are generated by combining at least two lipo amino fatty acids (LAFs) as hydrophobic cationizable motifs with hydrophilic cationizable aminoethylene units into novel sequence-defined molecules. The pH-dependent tunable polarity of the LAF is successfully implemented by inserting a central tertiary amine, which disrupts the hydrophobic character once protonated, resulting in pH-dependent structural and physical changes. This "molecular chameleon character" turns out to be advantageous for dynamic nucleic acid delivery via lipopolyplexes. By screening different topologies (blocks, bundles, T-shapes, U-shapes), LAF types, and LAF/aminoethylene ratios, highly potent pDNA, mRNA, and siRNA carriers are identified, which are up to several 100-fold more efficient than previous carrier generations and characterized by very fast transfection kinetics. mRNA lipopolyplexes maintain high transfection activity in cell culture even in the presence of ≥90% serum at an ultra-low mRNA dose of 3 picogram (≈2 nanoparticles/cell), and thus are comparable in potency to viral nanoparticles. Importantly, they show great in vivo performance with high expression levels especially in spleen, tumor, lungs, and liver upon intravenous administration of 1-3 µg luciferase-encoding mRNA in mice.