Correction of ADAMTS13 deficiency by in utero gene transfer of lentiviral vector encoding ADAMTS13 genes.

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (approximately 5-70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13(-/-) expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13(-/-) mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.

Enhancing the Cytotoxic Effects of PARP Inhibitors with DNA Demethylating Agents - A Potential Therapy for Cancer.

Poly (ADP-ribose) polymerase inhibitors (PARPis) are clinically effective predominantly for BRCA-mutant tumors. We introduce a mechanism-based strategy to enhance PARPi efficacy based on DNA damage-related binding between DNA methyltransferases (DNMTs) and PARP1. In acute myeloid leukemia (AML) and breast cancer cells, DNMT inhibitors (DNMTis) alone covalently bind DNMTs into DNA and increase PARP1 tightly bound into chromatin. Low doses of DNMTis plus PARPis, versus each drug alone, increase PARPi efficacy, increasing amplitude and retention of PARP1 directly at laser-induced DNA damage sites. This correlates with increased DNA damage, synergistic tumor cytotoxicity, blunting of self-renewal, and strong anti-tumor responses, in vivo in unfavorable AML subtypes and BRCA wild-type breast cancer cells. Our combinatorial approach introduces a strategy to enhance efficacy of PARPis in treating cancer.

Linked mechanical and biological aspects of remodeling in mouse pulmonary arteries with hypoxia-induced hypertension.

Right heart failure due to pulmonary hypertension causes significant morbidity and mortality. To study the linked vascular mechanical and biological changes that are induced by pulmonary hypertension, we mechanically tested isolated left main pulmonary arteries from mice exposed to chronic hypobaric hypoxia and performed histological assays on contralateral vessels. In isolated vessel tests, hypoxic vessels stretched less in response to pressure than controls at all pressure levels. Given the short length and large diameter of the pulmonary artery, the tangent Young's modulus could not be measured; instead, an effective elastic modulus was calculated that increased significantly with hypoxia [(280 kPa (SD 53) and 296 kPa (SD 50) for 10 and 15 days, respectively, vs. 222 kPa (SD 35) for control; P < 0.02)]. Hypoxic vessels also had higher damping coefficients [(0.063 (SD 0.017) and 0.054 (SD 0.014) for 10 and 15 days, respectively, vs. 0.033 (SD 0.016) for control; P < 0.002)], indicating increased energy dissipation. The increased stiffness with hypoxia correlated with an increase in collagen thickness (percent collagen multiplied by wall thickness) as well as the sum of elastin and collagen thicknesses measured histologically in the artery wall. These results highlight the mechanobiological changes in the pulmonary vasculature that occur in response to hypoxia-induced pulmonary hypertension. Furthermore, they demonstrate significant vascular mechanical and biological changes that would increase pulmonary vascular impedance, leading to right heart failure.