• ACVR1C (Activin receptor-like kinase 7, ALK7) is a type I receptor of the TGF-β superfamily. Activin E serves as its ligand, mediating activation of the Smad2/3 signaling pathway and regulating the expression of energy metabolism-associated genes. ALK7 modulates fat storage by suppressing lipolysis. Rare variant analysis using UK Biobank datasets shows that ALK7 loss-of-function is associated with favorable fat distribution patterns and lipid profiles. To date, small nucleic acid drugs—particularly siRNAs—represent the primary focus of ALK7-targeted drug development.
• Gene targeting strategy: The part of exon 2 to exon 9 of the mouse Alk7 gene, including 3’UTR, were replaced by P2A and the human full coding sequence (CDS), including human ALK7 3’UTR in B-hALK7 mice. The promoter and 5’UTR region of the mouse gene are retained. The human ALK7 expression is driven by the endogenous mouse Alk7 promoter, while the mouse Alk7 gene transcription and translation will be disrupted.
• mRNA expression analysis: Human ALK7 mRNA was exclusively detectable in B-hALK7 mice but not in wild-type mice. Mouse Alk7 mRNA was exclusively detectable in wild-type mice but not in B-hALK7 mice.
• The inhibitory efficiency of the small nucleic acid drugs: The human ALK7 in the siRNA group was significantly reduced compared to the control group.
• Application: This model is suitable for preclinical studies of obesity and small nucleic acid drug evaluation.

Gene targeting strategy for B-hALK7 mice. The part of exon 2 to exon 9 of the mouse Alk7 gene, including 3’UTR, were replaced by P2A and the human full coding sequence (CDS), including human ALK7 3’UTR in B-hALK7 mice. The promoter and 5’UTR region of the mouse gene are retained. The human ALK7 expression is driven by the endogenous mouse Alk7 promoter, while the mouse Alk7 gene transcription and translation will be disrupted.

Strain specific analysis of ALK7 mRNA expression in wild-type C57BL/6JNifdc mice and B-hALK7 mice by RT-PCR. Subcutaneous fat RNA was isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hALK7 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by RT-qPCR with human ALK7 primers. Human ALK7 mRNA was exclusively detectable in B-hALK7 mice but not in wild-type mice. Human sequences were confirmed via Sanger Sequencing.

Strain specific analysis of ALK7 mRNA expression in wild-type C57BL/6JNifdc mice and B-hALK7 mice by RT-qPCR. Perirenal fat, perigonadal fat, inguinal subcutaneous fat, and brown fat RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hALK7 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by RT-qPCR with mouse or human ALK7 primers. Human ALK7 mRNA was exclusively detectable in B-hALK7 mice but not in wild-type mice. Note: The male perirenal fat was not collected.

The inhibitory efficiency of the nucleic acid drugs against human ALK7 in heterozygous B-hALK7 mice. B-hALK7 mice (H/+) were randomly divided into two groups (male, 10 weeks old, n=5). The human ALK7 targeted nucleic acid drugs (synthesized according to patents), and PBS were administered to the mice individually. The nucleic acid drug was administered in the form of a PBS aqueous solution. The mice were sacrificed on day 14, and the adipose tissue was collected to detect the expression level of human ALK7 mRNA by qPCR. (A) The schematic diagram of experimental processing. (B) The expression of human ALK7 mRNA in the adipose tissues. Significance was determined by t-test, *P<0.05, **P<0.01, ***P<0.001. Values are expressed as mean ± SEM.