Single domain antibodies (sdAbs) correspond to the antigen-binding 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 ∼300 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 ∼1.8-fold higher liver LDLR 
protein levels. At 11 days, the equivalent values were 24 and 46% and ∼2.3-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.