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Although targeting TfR1 to deliver oligonucleotides to skeletal muscle has been demonstrated in rodents, effectiveness and pharmacokinetic/pharmacodynamic (PKPD) properties remained unknown in higher species. We developed antibody–oligonucleotide conjugates (AOCs) towards mice or monkeys utilizing anti-TfR1 monoclonal antibodies (αTfR1) conjugated to various classes of oligonucleotides (siRNA, ASOs and PMOs). αTfR1 AOCs delivered oligonucleotides to muscle tissue in both species. In mice, αTfR1 AOCs achieved a > 15-fold higher concentration to muscle tissue than unconjugated siRNA. A single dose of an αTfR1 conjugated to an siRNA against Ssb mRNA produced > 75% Ssb mRNA reduction in mice and monkeys, and mRNA silencing was greatest in skeletal and cardiac (striated) muscle with minimal to no activity in other major organs. In mice the EC50 for Ssb mRNA reduction in skeletal muscle was >75-fold less than in systemic tissues. Oligonucleotides conjugated to control antibodies or cholesterol produced no mRNA reduction or were 10-fold less potent, respectively. Tissue PKPD of AOCs demonstrated mRNA silencing activity primarily driven by receptor-mediated delivery in striated muscle for siRNA oligonucleotides. In mice, we show that AOC-mediated delivery is operable across various oligonucleotide modalities. AOC PKPD properties translated to higher species, providing promise for a new class of oligonucleotide therapeutics.

Bioactive peptides, including DNA-binding, endosomal release and cell targeting peptides, have been integrated into synthetic gene carriers to improve delivery efficiencies by enabling the vectors to overcome barriers to gene delivery. Our overall goal is to develop multifunctional, peptide-based polymers that incorporate motifs to condense DNA and facilitate sequential trafficking steps. One approach is to polymerize vinyl-terminated peptides by radical polymerization. In this work, cationic oligolysine peptides were designed to contain vinyl termini with internal reducible linkers. These peptides were copolymerized with HPMA to form biodegradable, DNA-condensing copolymers for gene delivery. The polymerization conditions were optimized by varying the initiator to monomer ratios, macromonomer to comonomer ratios, and reactant concentrations. The synthesized copolymers were shown to possess several important properties required for in vivo gene delivery applications, including (i) efficient DNA binding and condensation, (ii) the ability to stabilize particles against salt-induced aggregation, (iii) the ability to resist extracellular polyplex unpackaging, (iv) biocompatibility and the potential to be degraded into nontoxic components after cellular uptake, and (v) efficient delivery of plasmid to cultured cells.

Our report describes RAFT copolymerization of multiple species of active peptide monomers with N-(2-hydroxypropyl(methacrylamide) (HPMA) under aqueous conditions. Resulting statistical copolymers are narrowly disperse and with highly controlled molecular weight and composition. Side chain peptide-copolymers were synthesized using a DNA condensing peptide (K12), and an endosomal escape peptide (K6H5) that had been modified with an aminohexanoic linker and capped with methacrylamide vinyl on the NH2-terminus. Copolymers of HMPA-co-K12 and HPMA-co-K12-co-K6H5 efficiently condensed DNA into small particles that maintain size stability even in 150 mM salt solutions. With increasing peptide content, the peptide-based polymers demonstrated gene delivery efficiencies to cultured human cells that were comparable to linear polyethylenimine.