Efficient and safe correction of hemophilia A by lentiviral vector-transduced BOECs in an implantable device

Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch™) would rescue the bleeding phenotype of HA mice. To this end, BOECs from HA patients and healthy donors were isolated, expanded and transduced with an LV carrying FVIII driven by an endothelial-specific promoter employing GMP-like procedures. FVIII-corrected HA-BOECs were either directly transplanted into the peritoneal cavity or injected into a Cell Pouch™ implanted subcutaneously in NSG-HA mice. In both cases, FVIII secretion sufficient to improve the mouse bleeding phenotype. Indeed, FVIII-corrected HA-BOECs reached a relatively short-term clinically relevant engraftment being detected up to 16 weeks after transplantation, and their genomic integration profile did not show enrichment for oncogenes, confirming the process safety. Overall, this is the first pre-clinical study showing the safety and feasibility of transplantation of GMP-like produced LV-corrected BOECs within an implantable device for the long-term treatment of HA

EFFECT OF THE PHYTOESTROGEN DAIDZEIN ON TESTICULAR CELL FUNCTIONS

The soy bean isoflavone daidzein has weak estrogenic properties. Estrogens can disturb male reproduction by inhibiting steroidogenesis in Leydig cells. They may also cause decreasing sperm counts by affecting Sertoli cells which are crucial in spermatogenesis. We investigated the effect of estrogenic compounds on testicular cells in vitro. Cultures of rat Sertoli (SC) or Leydig (LC) cells were incubated with estrogens (nM to µM) for 3 d. Cytotoxicity, production of lactate and secretion of inhibin B (SC) or steroids (LC) were determined. 0.1-1 µM of estrogen did not alter any Sertoli cell parameter investigated. At higher concentrations (3-36 µM), only daidzein significantly reduced cell viability whereas ethinylestradiol significantly enhanced secretion of lactate and inhibin B. In contrast, bisphenol-A reduced inhibin levels. In Leydig cells, a dose-dependent decrease in progesterone and testosterone production was induced by nM of estradiol and ethinylestradiol, or µM of daidzein. Cell viability was not affected. Estrogen receptors are present in Sertoli and Leydig cells. Inhibition of LC testosterone synthesis by estradiol and ethinylestradiol was observed at nM concentrations and thus possibly be related to genomic effects. Daidzein acted only at µM levels. Here, a non-genomic mechanism, e.g. by competitive inhibition of enzymes involved in steroidogenesis is likely. A receptor-independent action may also be responsible for the observed effects of daidzein, ethinylestradiol, and bisphenol A on Sertoli cells. A daily intake of 1 mg/kg BW (= 3.9 µM) of daidzein is regarded as safe. This concentration, however, caused a significant reduction of androgens in our test system