Movement of the GAL locus to the nuclear periphery is part of a large-scale rearrangement of chromosome architecture induced by glucose withdrawal and is regulated by the activities of histone acetyltransferases and histone deacetylases.

Single domain antibodies (sdAbs) correspond to the antigenbinding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of ϳ300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and ϳ1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ϳ2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes.For over 30 years, a large number of clinical trials have firmly consolidated the importance of lowering LDL-cholesterol (LDLc) 3 in the prevention of cardiovascular diseases (CVD) and its associated devastating sequelae (1). Healthy diets and exercise are highly recommended to lower LDLc in patients with high baseline levels. However, many individuals, including those suffering from familial hypercholesterolemia (FH), cannot reach the recommended LDLc levels to prevent cardiovascular complications. With an overall incidence of ϳ1:200, FH is a common inherited disease that affects at least 30 million people, of whom Յ1% have been diagnosed. It is characterized by plasma LDLc levels greater that the 95th percentile, which result in tendon xanthomas, xanthelasmas, corneal arcus, and premature atherosclerosis, leading to premature ischemic vascular disease and mortality if left untreated. In most cases, FH subjects exhibit mutations in the LDL receptor (LDLR; 67%) and its ligand apolipoprotein B (apoB; 14%), hampering LDL clearance from the circulation (2). In 2003, merging biological studies with human genetics led to the discovery of PCSK9, the 9th and last member of the family of proprotein convertases related to subtilisin and kexin (3), and the demonstration that the PCSK9 gene represents the 3rd locus of autosomal domin...

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that binds and escorts the low density lipoprotein receptor (LDLR) into the lysosomal degradation pathway. Prescribed monoclonal antibodies (mAbs) against PCSK9 prevent its binding to the LDLR, and result in ~60% lower LDL cholesterol (LDLc) levels. Although efficient, mAbs are expensive. Hence other PCSK9 inhibitors are needed. For screening purpose, we developed C57BL/6J mice expressing the human PCSK9 gene under the control of its own promoter, but lacking endogenous mouse PCSK9. All lines recapitulate the endogenous PCSK9 expression pattern. The Tg2 line that expresses physiological levels of human PCSK9 (hPCSK9) was selected to characterize the inhibitory properties of a previously reported single domain antibody (sdAb), PKF8-mFc, which binds the C-terminal domain of PCSK9. Upon intraveinous injection of 10 mg/kg, PKF8-mFc and the mAb evolocumab neutralized ~50% and 100% of the hPCSK9 impact on total cholesterol (TC) levels, respectively, but PKF8-mFc had a more sustained effect. PKF8-mFc barely affected hPCSK9 levels, whereas evolocumab promoted a 4-fold increase 3 days post-injection, suggesting very different inhibitory mechanisms. The present study also shows that the new transgenic mice are well suited to screen a variety of hPCSK9 inhibitors.