Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells.

BACKGROUND: Epithelial sodium channel (ENaC) hyperactivity has been implicated in the pathogenesis of cystic fibrosis (CF) by dysregulation of fluid and electrolytes in the airways. In the present study, we show proof-of-principle for ENaC inhibition by lentiviral-mediated RNA interference. METHODS: Immortalized normal (H441) and CF mutant (CFBE) airway cells, and differentiated human bronchial epithelial cells in air liquid interface culture (HBEC-ALI) were transduced with a vesicular stomatitis virus G glycoprotein pseudotyped lentiviral (LV) vector expressing a short hairpin RNA (shRNA) targeting the α subunit of ENaC (ENaCα), and a marker gene. Efficacy of ENaCα down-regulation was assayed by the real-time polymerase chain reaction (PCR), membrane potential assay, western blotting, short-circuit currents and fluid absorption. Off-target effects were investigated by a lab-on-a-chip quantitative PCR array. RESULTS: Transduction to near one hundred percentage efficiency of H441, CFBE and HBEC-ALI was achieved by the addition of the LV vector before differentiation and polarization. Transduction resulted in the inhibition of ENaCα mRNA and antigen expression, and a proportional decrease in ENaC-dependent short circuit current and fluid transport. No effect on transepithelial resistance or cAMP-induced secretion responses was observed in HBEC-ALI. The production of interferon α and pro-inflammatory cytokine mRNA, indicating Toll-like receptor 3 or RNA-induced silencing complex mediated off-target effects, was not observed in HBEC-ALI transduced with this vector. CONCLUSIONS: We have established a generic method for studying the effect of RNA interference in HBEC-ALI using standard lentiviral vectors. Down-regulation of ENaCα by lentiviral shRNA expression vectors as shown in the absence off-target effects has potential therapeutic value in the treatment of cystic fibrosis.

Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype.

Pompe disease (acid alpha-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector-mediated expression of human acid alpha-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid alpha-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid alpha-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.

Hydroxyethyl starch-based preservation solutions enhance gene therapy vector delivery under hypothermic conditions.

Isolated liver perfusion offers a unique prospect for safe, effective targeting of gene therapies that can be directed against allograft rejection or recurrent diseases such as reinfection by hepatitis C virus (HCV). We aimed to examine the effect of organ preservation solutions on vector-based gene therapy delivery under hypothermic conditions. University of Wisconsin (UW) solution, histidine tryptophan ketoglutarate (HTK), EloHaes, sodium-poly(ethylene glycol)-UW solution [Institut Georges Lopez 1 solution (IGL-1)], and Dulbecco's modified Eagle's medium (DMEM) culture medium (control) were tested at 2 degrees C or 37 degrees C for lentiviral vector transduction efficiencies to the hepatoma cell line Huh-7 and primary human or mouse hepatocytes. Lentiviral vectors expressing short hairpin RNA were used to target HCV replication. With a potent short hairpin RNA vector, transductions were directly correlated to the therapeutic effect, with low transduction yielding low knockdown and vice versa. Green fluorescent protein (GFP) reporter gene expression was observed with vector incubation times as short as 10 minutes. The highest transductions were seen, after 2-hour 37 degrees C incubation, in UW (62% +/- 6 SEM); they were significantly higher than those in HTK (21% +/- 7 SEM). Neither adenosine nor glutathione, present in UW, provided any increase in transduction when supplemented to HTK, although the addition of hydroxyethyl starch (HES) significantly improved transductions. To rule out size exclusion as a mechanism of HES, IGL-1 was tested but did not result in better transductions than HTK or DMEM. When supplemented to UW, anionic compounds reduced transduction, and this indicated a charge interaction mechanism of HES. In conclusion, this study demonstrates that effective vector delivery can be achieved under conditions of hypothermic liver perfusion. UW provides superior transduction to hepatocytes over nonstarch solutions.

Simultaneous targeting of HCV replication and viral binding with a single lentiviral vector containing multiple RNA interference expression cassettes.

Chronic hepatitis C virus (HCV) infection has a major medical impact and current treatments are often unsuccessful. RNA interference represents a promising new approach to tackling this problem. The current study details the design and testing of self-inactivating lentiviral vectors (LV) delivering RNA interference to prevent HCV replication and infection. Vectors were constructed with single, double, and triple cassettes expressing short hairpin RNAs (shRNAs) simultaneously targeting two regions of the HCV 1b genome and the host cell receptor, CD81. The shRNAs directed against HCV IRES or NS5b regions were shown to be effective in inhibiting HCV replication in vitro (82 and 98%, respectively). No evidence of shRNA-related interferon production was observed. Vectors containing CD81 shRNA reduced cell surface expression up to 83% and reduced cell binding of HCV surface protein E2 up to 82% while not affecting levels of unrelated surface protein (Ber-EP4) or HCV replication. Double or triple shRNA vectors were independently effective in simultaneously reducing HCV replication, CD81 expression, and E2 binding. This study demonstrates lentiviral delivery of multiple shRNA, inhibiting HCV in a specific, IFN-independent, manner. The targeting of multiple viral and host cell elements simultaneously by RNAi could increase the potency of antiviral gene therapies.

Successful treatment of UGT1A1 deficiency in a rat model of Crigler-Najjar disease by intravenous administration of a liver-specific lentiviral vector.

Treatment of congenital and acquired liver disease is one of the main issues in the field of gene therapy. Self-inactivating lentiviral vectors have several potential advantages over alternative systems. We have constructed a self-inactivating lentiviral vector (LV-ALBUGT) that expresses the human bilirubin UDP-glucuronosyltransferase (UGT1A1) from a liver-specific promoter. UGT1A1 is involved in the clearance of heme metabolites in the liver. This enzyme is deficient in Crigler-Najjar disease, a recessive inherited disorder in humans characterized by chronic severe jaundice, i.e., high plasma bilirubin levels. Gunn rats suffer from the same defect and are used as an animal model of this disease. We have treated juvenile Gunn rats by single intravenous injection with the LV-ALBUGT vector. Over 1 year after treatment with the highest dose (5 x 10(8) transducing units), we observed a stable reduction of bilirubin levels to near normal levels and normal secretion of bilirubin conjugates in the bile, in contrast to untreated animals. In situ hybridization showed expression of the therapeutic gene in more than 30% of liver parenchymal cells. Thus, we demonstrate stable and complete clinical remission of a congenital metabolic liver disease in an animal model, after systemic administration of a therapeutic lentiviral vector.