Humanized KLKB1 provides a mouse model for evaluating the knockdown efficacy of RNAi therapies Free

Introduction: The KLKB1 gene encodes a glycoprotein that participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation, and inflammation. Plasma prekallikrein (PPK) is a glycoprotein predominantly synthesized in the liver. This protein is synthesized in the liver and circulates in the bloodstream. Factor XII converts prekallikrein into another protein called plasma kallikrein, which in turn helps activate additional factor XII. This product is used for pharmacodynamics and safety evaluation of Hereditary angioedema (HAE).

Methods: Targeting strategy: The exons 2-15 of mouse Klkb1 gene that encode the whole molecule (ATG to STOP codon), including 3'UTR were replaced by human counterparts in B-hKLKB1 mice. The promoter and 5'UTR region of the mouse gene were retained. The human KLKB1 expression was driven by endogenous mouse Klkb1 promoter, while mouse Klkb1 gene transcription and translation were disrupted.

mRNA expression analysis: Liver RNA was isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hKLKB1 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse and human KLKB1 primers.

Protein expression analysis: Plasma was collected from wild-type C57BL/6JNifdc mice (+/+) (male, n=3, 6-week-old) and homozygous B-hKLKB1 mice (H/H) (male, n=2, 6-week-old; female, n=3, 6-week-old). Expression level of human KLKB1 was analyzed by ELISA (Human Prekallikrein 1B ELISA Kit, Abcam, ab202405).

To evaluate the in vivo knockdown efficiency of RNAi therapy, heterozygous B-hKLKB1 mice were randomly divided into two groups (G1: n=3, 8 weeks old, Female; G2: n=3, 8 weeks old, Female). The human KLKB1 targeted nucleic acid drug were administered at a dose of 3 mpk subcutaneously. The mice were sacrificed on day 35.

Results: Protein expression analysis: Human Prekallikrein was exclusively detectable in homozygous B-hKLKB1 mice but not in wild-type mice.

mRNA expression analysis: Mouse Klkb1 mRNA was only detectable in wild-type mice. Human KLKB1 mRNA was exclusively detectable in homozygous B-hKLKB1 mice.

In vivo knockdown efficacy: Human KLKB1 targeted nucleic acid drugs was efficacious in B-hKLKB1 mice. The human KLKB1 in the treatment group was significantly reduced compared to the control group after administration.

Conclusions: B-hKLKB1 mice successfully express human KLKB1. They serve as a powerful in vivo platform to evaluate knockdown efficacy of RNAi therapy.