Overcoming Hemophilia A Gene Therapy Limitations with an Enhanced Function Factor VIII Variant.

Durable factor VIII (FVIII) expression that normalizes hemostasis is an unrealized goal of hemophilia A adeno-associated virus (AAV)-mediated gene therapy. Trials with initial normal FVIII activity observed unexplained year-over-year declines in expression while others reported low-level, stable FVIII expression inadequate to restore normal hemostasis. Here we demonstrate that mice recapitulate FVIII expression-level-dependent loss of plasma FVIII levels due to declines in vector copy number. We show that an enhanced function FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ), resistant to inactivation by protein C, normalizes hemostasis at below-normal expression levels without evidence of prothrombotic risk in hemophilia A mice. These data support that FVIII-QQ may restore normal FVIII function at low-levels of expression to permit durability using low AAV vector doses to minimize dose-dependent AAV toxicities. This work informs the mechanism of FVIII durability after AAV gene transfer and supports that incorporating the FVIII-QQ transgene may safely overcome current hemophilia A gene therapy limitations.

Macrophage and adipocyte interaction as a source of inflammation in kidney disease.

In obesity, adipose tissue derived inflammation is associated with unfavorable metabolic consequences. Uremic inflammation is prevalent and contributes to detrimental outcomes. However, the contribution of adipose tissue inflammation in uremia has not been characterized. We studied the contribution of adipose tissue to uremic inflammation in-vitro, in-vivo and in human samples. Exposure to uremic serum resulted in activation of inflammatory pathways including NFκB and HIF1, upregulation of inflammatory cytokines/chemokines and catabolism with lipolysis, and lactate production. Also, co-culture of adipocytes with macrophages primed by uremic serum resulted in higher inflammatory cytokine expression than adipocytes exposed only to uremic serum. Adipose tissue of end stage renal disease subjects revealed increased macrophage infiltration compared to controls after BMI stratification. Similarly, mice with kidney disease recapitulated the inflammatory state observed in uremic patients and additionally demonstrated increased peripheral monocytes and inflammatory polarization of adipose tissue macrophages (ATMS). In contrast, adipose tissue in uremic IL-6 knock out mice showed reduced ATMS density compared to uremic wild-type controls. Differences in ATMS density highlight the necessary role of IL-6 in macrophage infiltration in uremia. Uremia promotes changes in adipocytes and macrophages enhancing production of inflammatory cytokines. We demonstrate an interaction between uremic activated macrophages and adipose tissue that augments inflammation in uremia.

Cigarette Smoke Induces Metabolic Reprogramming of the Tumor Stroma in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is comprised of metabolically linked distinct compartments. Cancer-associated fibroblasts (CAF) and nonproliferative carcinoma cells display a glycolytic metabolism, while proliferative carcinoma cells rely on mitochondrial oxidative metabolism fueled by the catabolites provided by the adjacent CAFs. Metabolic coupling between these reprogrammed compartments contributes to HNSCC aggressiveness. In this study, we examined the effects of cigarette smoke-exposed CAFs on metabolic coupling and tumor aggressiveness of HNSCC. Cigarette smoke (CS) extract was generated by dissolving cigarette smoke in growth media. Fibroblasts were cultured in CS or control media. HNSCC cells were cocultured in vitro and coinjected in vivo with CS or control fibroblasts. We found that CS induced oxidative stress, glycolytic flux and MCT4 expression, and senescence in fibroblasts. MCT4 upregulation was critical for fibroblast viability under CS conditions. The effects of CS on fibroblasts were abrogated by antioxidant treatment. Coculture of carcinoma cells with CS fibroblasts induced metabolic coupling with upregulation of the marker of glycolysis MCT4 in fibroblasts and markers of mitochondrial metabolism MCT1 and TOMM20 in carcinoma cells. CS fibroblasts increased CCL2 expression and macrophage migration. Coculture with CS fibroblasts also increased two features of carcinoma cell aggressiveness: resistance to cell death and enhanced cell migration. Coinjection of carcinoma cells with CS fibroblasts generated larger tumors with reduced apoptosis than control coinjections, and upregulation of MCT4 by CS exposure was a driver of these effects. We demonstrate that a tumor microenvironment exposed to CS is sufficient to modulate metabolism and cancer aggressiveness in HNSCC. IMPLICATIONS: CS shifts cancer stroma toward glycolysis and induces head and neck cancer aggressiveness with a mitochondrial profile linked by catabolite transporters and oxidative stress. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/17/9/1893/F1.large.jpg.