Integrating HDAd5/35++ vectors as a new platform for HSC gene therapy of hemoglobinopathies

We generated an integrating, CD46-targeted, helper-dependent adenovirus HDAd5/35++ vector system for hematopoietic stem cell (HSC) gene therapy. The ∼12-kb transgene cassette included a β-globin locus control region (LCR)/promoter driven human γ-globin gene and an elongation factor alpha-1 (EF1α)-mgmtP140K expression cassette, which allows for drug-controlled increase of γ-globin-expressing erythrocytes. We transduced bone marrow lineage-depleted cells from human CD46-transgenic mice and transplanted them into lethally irradiated recipients. The percentage of γ-globin-positive cells in peripheral blood erythrocytes in primary and secondary transplant recipients was stable and greater than 90%. The γ-globin level was 10%–20% of adult mouse globin. Transgene integration, mediated by a hyperactive Sleeping Beauty SB100x transposase, was random, without a preference for genes. A second set of studies was performed with peripheral blood CD34+ cells from mobilized donors. 10 weeks after transplantation of transduced cells, human cells were harvested from the bone marrow and differentiated ex vivo into erythroid cells. Erythroid cells expressed γ-globin at a level of 20% of adult α-globin. Our studies suggest that HDAd35++ vectors allow for efficient transduction of long-term repopulating HSCs and high-level, almost pancellular γ-globin expression in erythrocytes. Furthermore, our HDAd5/35++ vectors have a larger insert capacity and a safer integration pattern than currently used lentivirus vectors

High and long-term von Willebrand factor expression after Sleeping Beauty transposon-mediated gene therapy in a mouse model of severe von Willebrand disease

BACKGROUND: Type 3 von Willebrand disease (VWD) is characterized by complete absence of von Willebrand factor (VWF). Current therapy is limited to treatment with exogenous VWF/FVIII products, which only provide a short-term solution. Gene therapy offers the potential for a long-term treatment for VWD. OBJECTIVES: To develop an integrative Sleeping Beauty (SB) transposon-mediated VWF gene transfer approach in a preclinical mouse model of severe VWD. METHODS: We established a robust platform for sustained transgene murine (m)VWF expression in the liver of Vwf(-/-) mice by combining a liver-specific promoter with a sandwich transposon design and the SB100X transposase via hydrodynamic gene delivery. RESULTS: The sandwich SB transposon was suitable to deliver the full-length mVWF cDNA (8.4 kb) and supported supra-physiological expression that remained stable for up to 1.5 year after gene transfer. The sandwich vector stayed episomal (~60 weeks) or integrated in the host genome, respectively in the absence or presence of the transposase. Transgene integration was confirmed using carbon tetrachloride-induced liver regeneration. Analysis of integration sites by high-throughput analysis revealed random integration of the sandwich vector. While the SB vector supported long-term expression of supraphysiological mVWF levels, the bleeding phenotype was not corrected in all mice. Long-term expression of VWF by hepatocytes resulted in relatively reduced amounts of high molecular weight multimers, potentially limiting its hemostatic efficacy. CONCLUSIONS: While this integrative platform for VWF gene transfer is an important milestone of VWD gene therapy, cell type specific targeting is yet to be solved

Antiinflammatory therapy with canakinumab for atherosclerotic disease

BACKGROUND: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. METHODS: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P=0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P=0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P=0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P=0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P=0.31). CONCLUSIONS: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. Copyright © 2017 Massachusetts Medical Society