The delivery of therapeutic oligonucleotides

Abstract

The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology.

Figure 1.

Tissue barriers to oligonucleotide delivery. Barriers for blood to parenchyma transfer are depicted. The star-shaped forms represent ‘free’ oligonucleotides or molecular scale oligonucleotide conjugates. The blue circles represent oligonucleotides incorporated in nanoparticles. Tissue parenchyma is represented as pink or tan (brain) coloration. (A) Blood brain barrier. The tightly apposed endothelial cells as well as pericytes and astrocyte processes present an essentially impenetrable barrier for both free oligonucleotides and oligonucleotides in nanoparticles. (B) Blood tissue barrier. In many tissues oligonucleotides can readily cross the endothelium by diffusion through paracellular routes. Permeation of nanoparticles is much more limited and may take place via transcytosis. (C) Blood liver barrier. The fenestrated endothelium in liver and spleen is easily permeated by both free oligonucleotides and nanoparticles. However, the liver kupffer cells avidly take up nanoparticles.

Figure 2.

Cellular uptake and intracellular trafficking of oligonucleotides. Oligonucleotides enter cells via several endocytotic pathways that vary in terms of their dependence on clathrin, caveolin or dynamin. These pathways all initially lead to the early/re-cycling endosome compartment; nonetheless molecules entering via different pathways can traffic to different downstream destinations. Most internalized oligonucleotide accumulates in late endosomes/multivesicular bodies (MVBs) and in lysosomes; however, some trafficking to other membrane bound compartments does occur. Oligonucleotides within endomembrane compartments are pharmacologically inert, but a very small portion of internalized oligonucleotide can spontaneously escape to the cytosol and nucleus. Intracellular trafficking is highly regulated by a large number of proteins and protein complexes. Thus the Rab family of GTPases regulates many aspects of trafficking and individual members serve as markers for distinct endomembrane compartments. The formation of MVBs is regulated by the multi-protein ESCRT complex that has recently been demonstrated to play a key role in the effectiveness of oligonucleotides. This complex also plays a role in exosome formation. The retromer complex may deliver oligonucleotides to the trans-Golgi instead of to lysosomes.

Figure 3.

Oligonucleotide delivery strategies. Several approaches to oligonucleotide delivery are depicted. (A) Antibody-oligonucleotide conjugate. (B) Polymer-oligonucleotide conjugate with PEGylation and targeting ligand. (C) Molecular scale ligand-oligonucleotide conjugate with triantennary carbohydrate ligand. (D) Lipid nanoparticle with PEGylation. (E) Gold nanoparticle with dense oligonucleotide coat. (F) DNA nanostructure with oligonucleotide and targeting ligand incorporated. Images are not to scale.