Lesson 6 – “Targeting” Ligand Approaches

Introduction

Today, conjugating ligands to ASOs seems trivial, but in 1990 when Mano Manoharan initiated the conjugation program at Ionis, it had never been done and a good many basic questions needed to be addressed.

Lipid conjugates 

They are widely studied as a general means to increase tissue uptake. Our experience has been negative, with some lipid related toxicities and PS ASO trapped in membranes.

Conclusions

At Ionis, we invented most of the medicinal chemistry of oligonucleotides. Though our major focus from day one was the 2’-position, we also focused on conjugation chemistry. The key decisions were where in an oligonucleotide to conjugate, what ligand to conjugate and what type of linker to use. We now know that we can conjugate ligands at many sites in an ASO, but most commonly, we use the 5’ or 3’ ends. We invented many types of linkers that vary in length, lipophilicity, charge, and stability in biologicals systems. The choice of which ligand to conjugate depends on what you are trying to achieve.

The most notable success to date has been to conjugate ligands to enhance delivery of ASOs to specific cells and tissues. To do that effectively, it is necessary to identify receptors that are highly expressed and can deliver high concentrations of ASOs to cells when the ligand binds to its receptor. GalNAc takes advantage of what hepatocytes do as one of their functions, i.e. clear specific chemicals from blood. GalNAc binds to a scavenger receptor expressed on hepatocytes, the asialoglycoprotein receptor to deliver ASOs to hepatocytes. By shifting a small fraction of plasma membrane bound PS ASO to bind to these receptors, GalNAc conjugation increased the potency of a PS ASO for hepatocyte targets by about 30-fold.

More surprising was the success of conjugating GLP-1 analogs to PS ASOs. This resulted in an enormous increase of PS ASO levels in beta cells of the pancreas, enabling targeting of these cells with PS ASOs for the first time. This was surprising because GLP-1 receptors are G-protein coupled receptors, with internalization being primarily a means of desensitization of the receptor via beta-arrestin.

Today, many programs are focused on using ligands to the transferrin receptor as a means of enhancing delivery to skeletal and cardiac muscle and across the blood-brain barrier.

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