Sato Y, Murase K, Kato J, Kobune M, Sato T, Kawano Y, et al. Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nat Biotechnol 2008;26:431–442. (Reprinted by permission from Macmillan Publishers Ltd: Nature Biotechnology, copyright 2008. www.nature.com/nbt/index.html.)
There are currently no approved antifibrotic therapies for liver cirrhosis. We used vitamin A-coupled liposomes to deliver small interfering RNA (siRNA) against gp46, the rat homolog of human heat shock protein 47, to hepatic stellate cells. Our approach exploits the key roles of these cells in both fibrogenesis as well as uptake and storage of vitamin A. Five treatments with the siRNA-bearing vitamin A-coupled liposomes almost completely resolved liver fibrosis and prolonged survival in rats with otherwise lethal dimethylnitrosamine-induced liver cirrhosis in a dose- and duration-dependent manner. Rescue was not related to off-target effects or associated with recruitment of innate immunity. Receptor-specific siRNA delivery was similarly effective in suppressing collagen secretion and treating fibrosis induced by CCl4 or bile duct ligation. The efficacy of the approach using both acute and chronic models of liver fibrosis suggests its therapeutic potential for reversing human liver cirrhosis.
Progressive liver fibrosis, or scarring of the liver, as the common consequence of all chronic liver diseases is a major medical and economic challenge, because there are no effective treatment options except liver transplantation. The current paradigm attributes this fibrogenic response to hepatic stellate cells (HSCs), which reside in the subendothelial space of Disse and are the major storage site for vitamin A in healthy humans. In response to injury, HSCs undergo an activation process in which they lose vitamin A, become highly proliferative, and synthesize abnormal extracellular matrix, including excessive collagen, various proteoglycans, and structural glycoproteins.1 This activation process and the deposition of extracellular matrix in the space of Disse are commonly considered to represent the key pathogenetic events in liver fibrosis.2
Previous studies showed that the collagen-specific chaperone heat shock protein 47 (HSP47)—also termed J6, gp46, collagen-binding 48 kDa protein (CB48), collagen binding protein 2 (CBP2), or colligin—plays an essential role in regulating collagen synthesis. It is constitutively expressed with collagens and binds to both helical and nonhelical forms of collagens.3 The link between Hsp47 and collagen production was demonstrated in Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of α(I) collagen and fibril structures in mesenchymal tissues.4 Simultaneous activation of both type-I and type-III collagens with Hsp47 expression was also reported in HSCs during carbon tetrachloride (CCl4)-induced liver fibrosis in rats.5 Conversely, the constitutive overexpression of Hsp47 promotes the secretion of collagens in vascular smooth muscle cells.6 As a molecular chaperone, HSP47 binds closely to procollagen in the endoplasmic reticulum, but dissociates from it in the Golgi complex to allow triple helix formation.7
Based on these properties Hsp47 has been suggested as plausible molecular target to interfere with fibrogenesis (Fig. 1). Initial proof-of-concept was obtained in experimental models, demonstrating that small interfering RNA (siRNA) targeting Hsp47 inhibited the expression of type-I collagen and the formation of scar tissue.8, 9
The study by Sato et al.10 targets siRNA directed against Hsp47 to HSCs. The authors used a key feature of HSCs, that is, uptake and storage of vitamin A, highlighted by Hans Popper more than 60 years ago.11 Sato et al. prepared retinol-coupled liposomes carrying siRNA against Hsp47 and demonstrated binding of retinol binding protein (RBP) to these liposomes (via gel filtration), receptor-mediated uptake of the RBP-coupled complexes by HSCs in vitro (via flow cytometry), and suppression of cellular collagen secretion (via Sirius red binding assays).
Using three different models of liver fibrosis (dimethylnitrosamine [DMN], CCl4, and bile duct ligation), the study assessed the antifibrotic properties of the siRNA-carrying liposomes in vivo at comparatively low doses of 0.75 mg/kg body weight. As shown by immunofluorescence and fluorescence-activated cell sorting analysis of isolated liver cells, the liposomes were predominantly taken up by HSCs in fibrotic livers. The effect of the siRNA on hepatic Hsp47 expression was confirmed by immunohistochemistry and immunoblots. The intervention decreased collagen deposition and induced apoptosis of HSCs, while animals were still being exposed to DMN, CCl4, or bile duct ligation. The liposomes improved liver function tests and prolonged survival of DMN-treated rats in a dose-dependent and duration-dependent manner. Importantly, the intervention did not induce hepatic expression of interferon-α, often associated with the use of siRNAs that contain the 5′ triphosphate of the T7-transcript, and did not increase serum levels of tumor necrosis factor-α and interleukin-12, indicating that the effects are not mediated by the innate immunity response.
Of note, the study indicates that activated HSCs, which lose their vitamin A stores during activation, continue to take up retinol effectively, most likely through vitamin A receptors. It has long been debated whether transfer of retinol from hepatocytes to stellate cells is receptor-driven or occurs via passive diffusion.12 After uptake of retinol, HSCs store it as retinyl esters or release it bound to RBP. In this study, RBP concentration-dependence and suppression by anti-RBP point to a receptor-mediated mechanism, although the identity of this receptor remains elusive. In contrast to the study by Sato et al., who used RBP-bound retinol, the major vitamin A source of HSCs is retinol bound to cellular retinol-binding protein I (CRBPI) released by hepatocytes, which obtain retinol from the human diet via the chylomicron pathway. CRBPI deficiency results in a marked reduction of retinyl ester accumulation in HSCs, which probably reflects an impaired delivery of retinol for esterification.12 Hence, it remains to be investigated whether the HSC specificity of the approach can be further improved by CRBP-coupled liposomes.
Although the title of the article implies resolution of liver cirrhosis, it has to be kept in mind that the fibrotic alterations in rodents are minor as compared to end-stage liver cirrhosis in humans,13 indicating the limitations of this preclinical study and its promise for clinical translation. At this stage it appears unlikely that a similar targeting complex will become a widely used therapy for liver cirrhosis in clinics, but the novel technological approach opens new avenues to modulate the deleterious fibrogenic response in chronic liver diseases.