Studies in gene therapy and drug targeting have brought to light the importance of identifying cellular and intracellular barriers to efficient delivery. Accordingly, a broad audience has been made aware in recent years of the characteristics of a typical trafficking pathway for many targeted therapeutics. Such a pathway is characterized by: receptor binding followed by cell entry via receptor-mediated endocytosis into clathrin-coated pits and vesicles, delivery to early endosomes, and passage through late endosomes/lysosomes where cargo degradation otherwise takes place [1
]. Ligand-receptor pairs, viruses and other pathogens, as well as non-viral gene delivery vectors are known to enter cells by such routes. If a targeted therapeutic, such as a gene delivery vector, is to impart therapeutic efficacy, however, the degradative pathway must somehow be avoided.
Endocytic pathways other than classical clathrin-mediated endocytosis targeted for the endosomal/lysosomal compartments have been better characterized in recent years. Such pathways may offer alternative uptake and trafficking routes for gene delivery vectors and targeted therapeutics that may avoid the barriers posed by the classical route. For example, the retrograde transport pathway, used by plant and bacterial toxins, facilitates endocytic trafficking from the cell surface to the Golgi, and from the Golgi to the endoplasmic reticulum (ER), in reverse of classical secretion [4
] (). These toxins can then make use of the cell’s own protein auditing system to become transported to the cytoplasm where the toxic activity can take place.
Figure 1 Retrograde transport of plant and bacterial toxins. Initial binding to a cell surface molecule triggers receptor-mediated endocytosis and delivery to early/sorting endosomes. Toxins may undergo delivery to the TGN from either early or late endosomes. (more ...)
Endocytosis via caveolae has been well-studied, and the route by which SV40 and similar pathogens utilize caveolar uptake for infection has been characterized in recent years [6
]. In this pathway, caveolar vesicles fuse with caveosomes, which facilitate prolonged survival of the pathogen in the cell before transit to the ER, from which nuclear entry can take place for viral replication ().
Figure 3 Trafficking via caveolae and caveosomes. SV40 bound to the cell surface can distribute to caveolae, which can pinch off to form a vesicle that is released from the cell surface and transported to Cav1-positive caveosomes. Alternatively, SV40 may directly (more ...)
Formation of replication-competent vacuoles inside cells is a strategy used by Brucella
and similar pathogens to gain long-term survival inside host cells [7
]. Such a pathway is typified by endocytosis into pathogen-containing vacuoles, delivery to and interaction with the ER, followed by formation of an ER-derived replicative organelle ().
Figure 4 Vacuole and replicating organelle formation. Membrane-bound Brucella is internalized into a Brucella-Containing Vacuole (BCV), which can either fuse with early/sorting endosomes, or undergo maturation, which entails transit to the ER. Interactions with (more ...)
Endocytic pathways may also lead to effective delivery and spread to neighboring cells. The secretion of exosomes likely enables prion proteins to be transmitted from cell to cell [8
Figure 2 Exosome-mediated spread of prion protein. Prion protein (PrP) may undergo either clathrin or caveolae -mediated endocytosis into early/sorting endosomes which can sort to multivesicular bodies (MVBs), into which intraluminal vesicles (ILVs) containing (more ...)
Why is studying these pathways important for therapeutic delivery? Delivery to the lysosomal compartment poses one major barrier to gene and drug delivery. The appeal of using viruses or viral components in targeted therapeutics is partly due to the capacity of endosomal escape, and thus avoidance of lysosomal degradation, by penetrating the membrane of the maturing vesicle before cargo delivery to the lysosome. Peptides derived from several types of pathogens have been used to accomplish the same [10
]. Such peptides are thought to change conformation in response to the acidifying environment of the endosomal lumen and as a result, interact with the endosomal membrane by forming pores or destabilizing the lipid bilayer, thus affording vesicle escape.
In the event that a gene delivery vector escapes the endocytic vesicle, cytosolic factors still pose additional barriers. The crowded cytosolic mileu can prevent rapid vector motility to the nucleus [11
] while cytosolic nucleases can degrade the DNA cargo [12
“Alternative” endocytic pathways such as those described above may contribute toward improvements in therapeutic delivery by facilitating: the avoidance of lysosomal delivery and degradation; enhanced delivery to a target organelle (such as the Golgi, ER, or nucleus) or compartment (such as the cytoplasm); and enhanced long-term therapy, such as the formation of an extranuclear replicating organelle.
It is clear that there is more than one route for entering a cell, and studies on a variety of pathogens show that alternative endocytic pathways have been cleverly hijacked to avoid a degradative fate and evade the cell’s defenses. Here we will examine some of these pathways, which may serve as possible routes for improving therapeutic delivery.