Conjugation Approaches for Peptide-Mediated Delivery of Oligonucleotides Therapeutics

Abstract

Oligonucleotide-based agents are versatile biomolecules that modulate gene expression. The last decade has seen the emergence of oligonucleotide-based tools for biochemical investigations. Importantly, several oligonucleotide-based drugs and vaccines are currently used for various therapeutic applications ranging from anti-inflammatory and anti-viral agents to those used in cardiovascular, ophthalmic, and neuro-muscular disorders. Despite a broad range of applications, achieving efficient oligonucleotide delivery remains a major limitation. A possible solution is to conjugate cellpenetrating peptides with oligonucleotides. This review provides an overview of chemical strategies used to synthesise peptide-oligonucleotide conjugates. The merits and liabilities of these strategies are discussed in the context of synthetic efficiency, and bio-reversible and -irreversible linkages.

Therapeutic Oligonucleotides: An Outlook on Chemical Strategies to Improve Endosomal Trafficking

“…Over the years, different types of ligation methods for tethering the oligonucleotides have been explored ( Figure 10 ) [ 202 , 203 ]. The most used non-cleavable (i.e., amide, triazole, and maleimide linkages) and chemically cleavable (i.e., disulfide and pH-sensitive linkages) linkers in oligonucleotide therapeutics are described in more details in this section.…”

“…The maleimide motif acts as a Michael acceptor and rapidly reacts with the free thiol group to form thiol-maleimide linkage. As this linkage can be prepared in an aqueous buffer without a catalyst, thiol-maleimide linkage has become a commonly used linkage to conjugate oligonucleotides with various ligands, such as antibodies [ 219 ], and cell penetrating peptides [ 203 , 220 , 221 ]. However, thiol-maleimide linkages are susceptible to in vivo reduction, resulting in premature release of oligonucleotides from the ligand [ 222 , 223 ].…”

“…However, thiol-maleimide linkages are susceptible to in vivo reduction, resulting in premature release of oligonucleotides from the ligand [ 222 , 223 ]. The use of retro-Michael ring opening of thiol-maleimide linkage is an alternative approach to overcome the potential reduction possibilities [ 203 , 224 , 225 ]. Anti-CD71 Fab antibody–siRNA conjugates (ARCs) using thiol-maleimide linkage enable to achieve significant gene silencing in muscle tissues [ 226 ].…”

Iron uptake pathway of Escherichia coli as an entry route for peptide nucleic acids conjugated with a siderophore mimic

“…Another factor limiting PNA uptake in gram-negative bacteria is the lipopolysaccharide (LPS) layer, which forms a negatively charged structural barrier protecting the inner leaflet of the outer membrane and preventing passive diffusion of hydrophobic molecules ( Good et al, 2000 ; Bertani and Ruiz, 2018 ). Therefore, the conjugation of PNA oligomers to carriers that would enable PNA transport across the membrane has been investigated ( Lu et al, 2010 ; Volpi et al, 2021 ; Patil, 2022 ; Patil et al, 2022 ). One strategy is to use cell-penetrating peptides (CPPs), such as (KFF) 3 K, TAT or (RXR) 4 XB ( Bendifallah et al, 2006 ; Barkowsky et al, 2019 , 2022 ; Patil et al, 2019 ; Wojciechowska et al, 2020 ; Yavari et al, 2021 ; Tsai et al, 2023 ).…”

An Efficient Approach for the Design and Synthesis of Antimicrobial Peptide-Peptide Nucleic Acid Conjugates

“…Importantly, conjugation of PNAs with structurally complex peptides such as the polymyxins ( Velkov et al, 2013 ) and cathelicidin ( Dürr et al, 2006 ) remains an unexplored terrain ( Soudah et al, 2019 ). Moreover, peptide conjugations through traditional disulfide and maleimide linkages require orthogonal protecting groups as well as a multistep synthesis strategy ( Patil, 2021 ). Previously, we developed a methodology for cysteine-cyanobenzothiazole (Cys-CBT) mediated peptide-PNA conjugation that enables facile synthesis and improved intracellular stability as compared to other strategies ( Patil et al, 2019 ).…”