hC5(Plus)

Fig.1 Detection of C5 expression in liver by RT-PCR. Mouse C5 (Hc) mRNA (206 bp) was detectable only in WT C57BL/6 mice. Human C5 mRNA (165 bp) was detectable only in HO hC5 knockin mice but not in WT mice.

Wild type: only one band at 206 bp with primers F1/R1(mHc);

Homozygous: only one band at 165 bp with primers F2/R2(hC5).

Abbr. M, DNA marker; HO, homozygous; WT, wild type.

Fig.2 Detection of human C5(A) and mouse C5(B) expression in serum by ELISA (n=3, female, 6-week-old). 

Abbr.  HO, homozygous; WT, wild type; N.D. not detected.

Fig.3 Analysis of the alternative pathway activity. Serum was collected and evaluated with the complement kit. WT C57BL/6 mice exhibited robust alternative complement pathway activity, while hC5 mice showed significantly reduced AP activity, indicating that human C5 expression does not fully restore the mouse alternative complement pathway. The hC5(Plus) line displayed AP activity comparable to wild-type levels, suggesting improved functional complement restoration in this variant line.

Abbr.  HO, homozygous; WT, wild type

Fig.4 Detection of human C5 expression in serum of hC5 and hC5(Plus) mice by ELISA following Cemdisiran (ALN-CC5) treatment. Cemdisiran (ALN-CC5) effectively depleted human C5 protein in both hC5 and hC5(Plus) mice, with hC5 mice showing complete C5 suppression from Day 7 onwards, while hC5(Plus) mice maintained stable baseline C5 levels under vehicle treatment and exhibited sustained C5 reduction upon Cemdisiran administration. hC5 (n=5, female, 11 week-old ) and hC5(Plus) mice  (n=3, male, 10 week-old )  were administered vehicle (i.v.) or Cemdisiran (3 mg/kg, s.c.) on Day 0, and serum human C5 concentrations were measured at indicated time points (D0, D7, D14, D21, D28) by ELISA (Human C5 Elisa Kit,). Data are presented as mean ± SEM. 

Abbr.  HO, homozygous; WT, wild type; N.D. not detected；i.v., intravenous; s.c., subcutaneous; Conc., concentration.