IntroductionGlycogenosis type II (Pompe disease, acid maltase deficiency; Online Mendelian Inheritance in Man no. 232300) is an autosomal recessive lysosomal storage disorder caused by acid ␣-glucosidase (GAA) deficiency. The disease is characterized by glycogen storage in liver, spleen, kidney, brain, and endothelial cells and most prominently in skeletal, heart, and smooth muscles. Symptoms arise from muscular weakness and wasting. Infants with complete enzyme deficiency present shortly after birth, lose all muscle strength within 8 months, and succumb to hypertrophic cardiomyopathy and respiratory failure in the first year of life. 1 Children and adults with residual GAA activity show a more protracted course and may become wheelchair bound, dependent on artificial ventilation, and have a shortened life expectancy. Presently, enzyme replacement therapy (ERT) based on intravenous infusion of recombinant human ␣-glucosidase, taken up by mannose 6-phosphate receptor mediated endocytosis, 2,3 is a major therapeutic advance that prolongs the life of affected infants but does not guarantee long-term symptom-free survival, requires biweekly administration, and may induce immune responses to the recombinant protein.As an alternative to ERT, in vivo gene therapy mediated by adenoviral vectors and adeno-associated virus vectors (AAVs) has been investigated in a mouse model of Pompe disease. [4][5][6] However, long-term efficacy can be significantly hampered by antibody formation, 7,8 and adverse immune responses to the vector has been observed after adenoviral and AAV gene therapy in patients. 9,10 For treatment of patients with other lysosomal enzyme deficiencies, allogeneic hematopoietic stem cell (HSC) transplantation has been proposed. 11 HSC transplantation proved effective in ameliorating the neurologic symptoms in murine globoid cell leukodystrophy and human patients 12,13 as well as in mucopolysaccharidosis I (Hurler syndrome). 14,15 Other lysosomal storage disorders such as metachromatic leukodystrophy (MLD) may require higher enzyme levels than provided by HSC transplantation; lentiviral (LV) vector-mediated overexpression of aryl-sulfatase A in HSCs effectively reversed the neuropathologic phenotype in the mouse model. 16 In addition, LV-mediated clinical gene therapy in trial phase of X-linked adrenoleukodystrophy halted progressive cerebral demyelination in 2 patients. 17 Recently, HSC transplantation was shown to promote immune tolerance to ERT in the Pompe mouse model. 18 The use of gene-modified autologous HSCs also overcomes the profound conditioning and immune barriers associated with allogeneic transplantation.The few attempts of HSC transplantation for Pompe disease have not met with success. 19 GAA levels, if any, are low in hematopoietic cells in mice, 18 and allogeneic transplantation is not an obvious treatment. Therefore, high-level vector-driven ectopic expression of the enzyme in hematopoietic cells would be required to accomplish efficacy. We tested the hypothesis that ex vivo LV For persona...

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.

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.

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.