Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotide using FORCE conjugation in mdx mice

Abstract

Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE–M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE–M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE–M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.

RNAi in cell nuclei: potential for a new layer of biological regulation and a new strategy for therapeutic discovery

“…Delivery of ASOs and dsRNAs to the liver is well-established, but progress is also being made with delivery to the central nervous system (Bennett 2019;Bennett et al 2019), the eye (Garanto 2022), lungs (Shin et al 2022), muscle (Desjardins et al 2022), and other organs. Applications range from N=1 therapies for ultra-rare diseases (Kim et al 2019;Dhuri et al 2020) to the potential for broad use of dsRNAs to be used to reduce high cholesterol levels (Migliorati et al 2022).…”

A Review of Protein- and Peptide-Based Chemical Conjugates: Past, Present, and Future

“…In the case of Avidity, their AOC programs have opted for a noncleavable linker to ensure maximal delivery to cells [ 57 ]. On the other hand, as part of their FORCE™ platform, Dyne uses a well-known cleavable ADC linker val-cit, in order to achieve maximal modification of the target [ 57 , 58 ]. This linker is stable in plasma but will be enzymatically cleaved in the endosomal compartment to enable release of the oligonucleotide payload in the cytosol.…”

Drug Metabolism and Pharmacokinetics of Antisense Oligonucleotide Therapeutics: Typical Profiles, Evaluation Approaches, and Points to Consider Compared with Small Molecule Drugs

“…Other examples of specific receptor targeting include the conjugation of glucagon-like peptide-1 receptor (GLP1R) agonist ligands as carrier peptides for targeted delivery of ASOs to pancreatic β cells [ 66 ]; a neurotensin receptor–ligand system for targeted delivery of ASOs to the CNS [ 67 ]; and transferrin receptor 1 (TfR1)-targeted delivery of ASOs to muscular tissues by conjugating them to a TfR1-binding fragment [ 68 ]. Recently, growing attention has been directed toward the conjugation of ASOs to lipid/fatty acid ligands.…”