1.4 Cell-specific gene transfer
1.4.3 Targeted gene transfer to hepatocytes
The first demonstration of the potential for hepatocyte-specific gene transfer via ASGP-R- mediated endocytosis emerged in the 1980s. The pioneering study by Wu and Wu entailed the covalent attachment of asialoorosomucoid, a natural ligand to the ASGP-R, to poly-L- lysine, for the introduction of the chloramphenicol acetyltransferase (CAT) reporter gene into liver parenchymal cells in vitro and in vivo (Grove and Wu, 1998; Wu and Wu, 1998).
However the sophisticated procedure involved in the preparation of this carrier (Ren et al., 2001) and its reproducibility (Kawakami et al., 1998) were problematic. Therefore
subsequent attempts at directing genes to hepatocytes by receptor-mediation employed more promising non-viral gene transfer agents, notably the cationic liposomes.
1.4.3.1 Liposome modification with ASGP-R-specific ligands
1.4.3.1.1 Asialoglycoproteins (ASGPs)
The asialoglycoproteins are a class of endogenous glycoproteins from which the terminal sialic acid residue has been enzymatically removed. Asialoorosomucoid (AOM), asialofetuin (AF), asialolactoferrin, asialotransferrin and asialoceruloplasmin have been appended to liposomal carriers in order to direct hepatocellular recognition (Pathak et al., 2008).
Singh and colleagues (2010) made use of the high affinity streptavidin-biotin interaction to attach AOM to a cationic liposome. This multicomponent lipoplex, illustrated in Figure 1.10,
23 not only mediated high transfection activity in cultured hepatocytes, but also showed
favourable size, charge and cytotoxicity profiles for potential in vivo application.
Figure 1.10: Schematic representation of the hepatocyte-specific modular complex formed between plasmid DNA, biotinylated liposomes, streptavidin and dibiotinylated AOM. B:
biotin, S: streptavidin, AOM: asialoorosomucoid, GAL: galactose residue, R:
asialoglycoprotein receptor (Singh et al., 2010).
In a deviation from the use of unmodified ASGPs as targeting ligands, Singh and Ariatti (2003) designed a hepatotropic transfecting complex assembled from the spontaneous electrostatic interactions between activated cationic liposomes, pRSVL plasmid DNA and a carbodiimide-cationised derivative of AOM. In transfection studies employing the human hepatoma cell line, HepG2, the ternary complex demonstrated luciferase activity four times higher than complexes assembled from non-cationised AOM.
The inclusion of asialofetuin as a targeting component of liposomal gene transfer systems is relatively prominent in the literature. Early work by Hara et al. (1995) entailed the
application of both detergent removal and freeze thaw procedures to simultaneously permit the encapsulation of pSV2CAT DNA within, and its binding to the membranes of AF-
labelled cationic liposomes. Arangoa and coworkers (2003) attempted to develop this strategy by designing a serum-tolerant hepatocyte-directed lipoplex. The reporter plasmid was
condensed via a cationic peptide, protamine, and subsequently complexed with AF-modified DOTAP/Chol liposomes. The ASGP-R-affinity of AF, combined with the nuclear localisation
24 potential of protamine, afforded successful introduction of reporter genes into liver
parenchymal cells following systemic administration in mice, without inducing organ damage. More recently, efforts by Tros de Ilarduya (2010) produced high levels of transfection in HepG2 cells, having improved the serum tolerance of AF-modified DOTAP/Chol liposomes. Furthermore, studies by Dasi et al. (2001) adds credence to the feasibility of AF-modified liposomes as gene transfer agents to cells of hepatic origin, as such a vector facilitated the introduction and sustained expression of the medically significant α1- antitrypsin gene in an animal model.
1.4.3.1.2 Galactose
While ASGPs are proven to facilitate effective targeting of liver parenchymal cells, their application as components of non-viral vectors is limited by the high cost and tedious procedures required for their purification (Hwang et al., 2001). Due to the fact that the terminal galactose and GalNAc residues of ASGPs mediate their recognition by binding to the ASGP-R; these monosaccharides and other galactose-terminated compounds, such as lactose (Watanabe et al., 2007), lac-BSA (Pathak et al., 2008) and lactobionic acid (Yu et al., 2007), were investigated as alternative hepatocyte-targeting moieties. In recent years,
structure-affinity studies involving the human ASGP-R have established that GalNAc binds with 50 fold greater affinity than galactose (Khorev, 2007; Westerlind et al., 2004). However, it is the galactose moiety that has received the most attention as a hepatocyte-specific homing device for gene transfer applications (Pathak et al., 2008).
Several liposomal gene transfer approaches employ galacto-entities that are displayed from the surface of the liposome, by way of appending the monosaccharide to a membrane- compatible lipid that permits its stable anchorage to the bilayer. Cholβgal (Figure 1.11a), composed of a β-D-galactose residue attached via a glycosidic bond to a cholesterol anchor, is among the simplest glycolipids (Singh et al., 2007). However, in the interest of ensuring optimal interaction between the liposome-appended targeting moiety and the ASGP-R, glycolipid designs have developed to include lipids bearing both cyclic and open galacto- headgroups (Mukthavaram et al., 2009), multifunctional lipids (Kawakami et al., 2000), variation in the length of the spacer between the lipophilic anchor and monosaccharide
25 (Kawakami et al., 1998), as well as the number of galactose residues tethered to a single lipid (Jiang et al., 2008; Tang et al., 2007). In fact, several synthetic glycolipids (Figure 1.11) have yielded encouraging results in vitro and in vivo.
As an example, Kawakami and coworkers (1998) evaluated the efficacy of liposomes prepared from DC-Chol, DOPE and a newly synthesised cationic glycolipid Gal-C4-Chol (Figure 1.11b). This vector system exhibited markedly higher transfection activity (as measured by reporter gene expression and [32P] DNA uptake) in HepG2 cells, than cationic liposomes formulated without the glycolipid. Furthermore, gene expression using the targeted formulation was markedly reduced after exposing the cells to 20 mM galactose; which
affirmed ASGP-R- mediated vector internalisation by the liver parenchymal cells. In a related study, Gal-C4-Chol/DOTMA/Chol liposomes achieved hepatocyte-specific introduction of the luciferase reporter gene following intraportal administration in mice (Kawakami et al., 2000).
Importantly, several groups have demonstrated that although the inclusion of a prominently displayed hepatocyte-targeting moiety is essential, physical features such as galactose density, lipid composition, size, stability, and charge ratio of the lipoplexes must be optimised in order to develop effective liver-directed, cationic liposomal gene transfer systems (Fumoto et al., 2004; Kawakami et al., 2000; Managit et al., 2005).
26
O
OH OH OH HO
O
O
OH OH OH HO
S NH
NH2
NH O
O
O O N N
O OH OH HO
O
H H
O OH
OH OH OH HO
O O
O O O
O O S
OH OH HO
OH
S O
OH OH HO
OH
S O
OH OH HO
OH
S O OH
O OH HO
S O OH
O OH HO
O
H H O 6 O
Figure 1.11: Examples of glycolipids used in hepatotropic liposome formulations, a)
cholesteryl-β-D-galactopyranoside (Cholβgal) (Singh et al., 2007); b) cholesten-5-yloxy-N-(- b)
c)
d) a)
27 4-((1-imino-c-β-D-thiogalactosyl-ethyl) amino) butyl) formamide (Gal-C4-Chol) (Shigeta et al., 2007); c) (5-cholestan-3β-yl)-1-[2-(lactobionyl amido) ethylamido] formate (CHE-LA) (Yu et al., 2007); and d) penta-antennary thiogalactoside L-II (Jiang et al., 2008).