Erwei Song

RNA interference targeting Fas protects mice from fulminant hepatitis

RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. However, its potential to treat or prevent disease remains unproven. Fas-mediated apoptosis is implicated in a broad spectrum of liver diseases, where inhibiting hepatocyte death is life-saving. We investigated the in vivo silencing effect of small interfering RNA (siRNA) duplexes targeting the gene Fas (also known as Tnfrsf6), encoding the Fas receptor, to protect mice from liver failure and fibrosis in two models of autoimmune hepatitis. Intravenous injection of Fas siRNA specifically reduced Fas mRNA levels and expression of Fas protein in mouse hepatocytes, and the effects persisted without diminution for 10 days. Hepatocytes isolated from mice treated with Fas siRNA were resistant to apoptosis when exposed to Fas-specific antibody or co-cultured with concanavalin A (ConA)-stimulated hepatic mononuclear cells. Treatment with Fas siRNA 2 days before ConA challenge abrogated hepatocyte necrosis and inflammatory infiltration and markedly reduced serum concentrations of transaminases. Administering Fas siRNA beginning one week after initiating weekly ConA injections protected mice from liver fibrosis. In a more fulminant hepatitis induced by injecting agonistic Fas-specific antibody, 82% of mice treated with siRNA that effectively silenced Fas survived for 10 days of observation, whereas all control mice died within 3 days. Silencing Fas expression with RNAi holds therapeutic promise to prevent liver injury by protecting hepatocytes from cytotoxicity.

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors

Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

Sustained Small Interfering RNA-Mediated Human Immunodeficiency Virus Type 1 Inhibition in Primary Macrophages

ABSTRACT Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

Interfering with disease: opportunities and roadblocks to harnessing RNA interference

n Abstractn n RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA. Short double-stranded RNAs, known as small interfering RNAs (siRNA), are incorporated into an RNA-induced silencing complex that directs degradation of RNA containing a homologous sequence. RNAi has been shown to work in mammalian cells, and can inhibit viral infection and control tumor cell growth in vitro. Recently, it has been shown that intravenous injection of siRNA or of plasmids expressing sequences processed to siRNA can protect mice from autoimmune and viral hepatitis. RNAi could provide an exciting new therapeutic modality for treating infection, cancer, neurodegenerative disease and other illnesses.n n

Inhibition of matrix metalloproteinases during chronic allograft nephropathy in rats.

Background. Chronic allograft nephropathy (CAN) belongs to the major causes of long-term kidney allograft failure. One of the histologic hallmarks of CAN is interstitial fibrosis, influenced by matrix metalloproteinases (MMPs) that are controlling extracellular matrix (ECM) degradation. Whether MMPs affect the development and progression of CAN is not clear so far. To analyze the role of MMPs in CAN, we investigated the effects of an early and a late application of BAY 12–9566, an inhibitor of MMP-2, -3, and -9 on the development and progression of CAN in a rat kidney-transplantation model. Methods. Fisher kidneys were orthotopically transplanted into Lewis recipients that were treated with BAY 12–9566 (15 mg/kg per day) or vehicle either for the first 10 days after transplantation (early treatment) or from week 12 to week 20 after transplantation (late treatment). Proteinuria was analyzed every 4 weeks up to week 20 after transplantation when kidney grafts were removed for further analysis. Results. Early MMP-inhibition resulted in a significantly reduced 24-hour protein excretion that was paralleled by a lower grade of CAN after 20 weeks. However, late MMP inhibition starting at week 12 after transplantation resulted in significantly higher proteinuria and a higher grade of CAN as compared with controls. Furthermore, transforming growth factor-&bgr; and platelet-derived growth factor-B chain mRNA levels were significantly increased in these animals. Conclusions. Inhibition of MMPs early after transplantation reduced the development and progression of CAN but promoted CAN if initiated at later stages. Thus, MMPs are involved in the development and progression of CAN.

Opposite effects of testosterone and estrogens on chronic allograft nephropathy.

Abstract In the present study we investigated whether donor gender of the effects of sex hormones play the greater role in the development of chronic allograft nephropathy. Kidneys of male and female Fisher rats were orthotopically transplanted into castrated male Lewis recipients. Animals were treated with testosterone, estradiol, or vehicle and the kidneys were harvested 20 weeks after transplantation for histological, immunohistological, and molecular analysis. Testosterone treatment resulted in increased proteinuria and profound glomerulo‐sclerosis, irrespective of donor gender. In addition, mRNA levels of transforming growth factor‐β1 (TGF‐β1) and platelet‐derived growth factor‐A and B (PDGF‐A and B) chains were enhanced in these allografts. Estradiol reduced glomerulosclerosis and mononuclear cell infiltration in allografts of both genders that paralleled a decreased mRNA expression of TGF‐β1, PDGF‐A and B. No donor gender‐related differences were noted in vehicle‐treated animals. Our findings demonstrate that sex hormones rather than donor gender have a significant impact on chronic allograft nephropathy.