hAGT/hREN(2)

Fig.1 Detection of AGT(A) and REN(B) expression in liver by RT-PCR. 

Wild type: only one band at 232 bp with primers F1/R1(mAgt); only one band at 235 bp with primers F3/R4(mRen);

Homozygous: only one band at 360 bp with primers F2/R2(hAGT); only one band at 202 bp with primers F4/R4(hREN)

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

Fig.2 Detection of hAGT and mAGT expression in plasma by ELISA (male, n=3, 7-8wks). 

Abbr. HO, homozygous; WT, wild type.

Fig.3 Detection of hREN expression in plasma by ELISA (male, n=3, 7-8wks). 

Abbr. HO, homozygous; WT, wild type.

Fig.4 Detection of mANG2 expression in plasma by ELISA (male, n=3, 7-8wks). 

Abbr. HO, homozygous; WT, wild type.

Fig.5 Detection of human AGT and hREN levels in serum from male hAGT/hREN(2) mice. In hAGT/hREN(2) double knock-in mice, serum human AGT levels remain stable across different ages, whereas serum human REN levels decline significantly after 11 weeks of age. Serum was collected weekly for the detection of hAGT levels using the ELISA kit and hREN levels using the ELISA kit. (Mean±SEM, Unpaired t-test, **p<0.01, ****p<0.0001; ns, no significance).

Abbr. HO, homozygous.

Fig.6 Detection of human AGT reduction in serum from male hAGT/hREN(2) mice. Zilebesiran markedly suppressed serum human AGT levels in hAGT/hREN(2) mice, maintaining a sustained reduction over a 6-week period. Male hAGT/hREN(2) mice (15 weeks old) were assigned to two groups (n=9 per group). Mice in Group 1 received a single subcutaneous injection of PBS, while mice in Group 2 were administered a single subcutaneous dose of Zilebesiran. Serum was collected weekly for the detection of hAGT levels using the ELISA kit. (Mean±SEM, Unpaired t-test, ***p<0.001, ****p<0.0001; ns, no significance).

Abbr. HO, homozygous.

Fig.7 Detection of human REN levels in serum from male hAGT/hREN(2) mice. Zilebesiran induced a compensatory elevation of serum human REN levels in hAGT/hREN(2) mice. Male hAGT/hREN(2) mice (15 weeks old) were assigned to two groups (n=9 per group). Mice in Group 1 received a single subcutaneous injection of PBS, while mice in Group 2 were administered a single subcutaneous dose of Zilebesiran. Serum was collected weekly for the detection of hREN levels using the ELISA kit. (Mean±SEM, Unpaired t-test, **p<0.01, ***p<0.001, ****p<0.0001; ns, no significance).

Abbr. HO, homozygous.

Fig.8 Detection of AGT expression in liver by qPCR. Zilebesiran significantly knocked down hepatic human AGT mRNA expression in hAGT/hREN(2) knock-in mice. Male hAGT/hREN(2) mice (15 weeks old) were assigned to two groups (n=9 per group). Mice in Group 1 received a single subcutaneous injection of PBS, while mice in Group 2 were administered a single subcutaneous dose of Zilebesiran (15 mg/kg, MCE, HY-145649). Liver RNA was extracted every two weeks (n=3/group), then cDNA libraries were synthesized by reverse transcription, followed by qPCR with human AGT and mouse Gapdh primers. Relative expression represents the human AGT mRNA level relative to its average expression in Group 1 mice at 2 weeks post-treatment. (Mean±SEM, Unpaired t-test, *p<0.05, **p<0.01; ns, no significance). 

Abbr. HO, homozygous.