• Background: Alpha-1 antitrypsin (AAT), or SERPINA1, is a crucial serine proteinase inhibitor produced by hepatocytes and secreted into the bloodstream. It primarily functions in the lungs, protecting elastic tissue from neutrophil elastase and inhibiting other enzymes like trypsin, plasmin, and thrombin. This activity helps safeguard normal cells and organs from proteolytic harm, suppresses infections and inflammation, and maintains internal balance.
• Targeting strategy: The  exons 2-5 of human SERPINA1*E342K, including 3’UTR that driven by Alb promoter are inserted into mouse Hipp11 (H11) locus in B-hSERPINA1*E342K mice.
• Validation: Human SERPINA1 protein was exclusively detectable in homozygous B-hSERPINA1*E342K mice. Human SERPINA1 mRNA was exclusively detectable in homozygous B-hSERPINA1*E342K mice.
• Human SERPINA1 protein was significantly decreased after Fazirsiran analog dosage and B-hSERPINA1*E342K mice can be used in small nucleic acid drug screening research.
• Application: Over 100 mutations in the SERPINA1 gene have been identified, with Pi*Z (E342K) and Pi*S being the most common. The ZZ homozygous mutation is particularly associated with alpha-1 antitrypsin deficiency (AATD) and chronic obstructive pulmonary disease (COPD). Additionally, abnormal, insoluble polymeric aggregates of AAT (Z-AAT) accumulate in hepatocytes, leading to cell damage and potentially resulting in severe liver disease and failure

Gene targeting strategy for B-hSERPINA1*E342K mice. The  exons 2-5 of human SERPINA1*E342K, including 3’UTR that driven by Alb promoter are inserted into mouse Hipp11 (H11) locus in B-hSERPINA1*E342K mice.

Strain specific analysis of SERPINA1 mRNA expression in wild-type C57BL/6 mice and B-hSERPINA1* E342K mice by RT-PCR. Liver RNA was isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hSERPINA1*E342K mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with human SERPINA1 primers. Human SERPINA1 mRNA was detectable only in homozygous B-hSERPINA1*E342K mice but not in wild-type mice.

Strain specific SERPINA1 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hSERPINA1*E342K mice by ELISA. Plasma was collected from wild-type C57BL/6JNifdc mice (+/+) (male, n=3, 6-week-old) and homozygous B-hSERPINA1*E342K mice (H/H) (male, n=3, 6-week-old). Expression level of human SERPINA1 was analyzed by ELISA (anti-human alpha 1 Antitrypsin ELISA kit: Abcam, ab108799). Human SERPINA1 was exclusively detectable in homozygous B-hSERPINA1*E342K mice (n=3). Values are expressed as mean ± SEM.

Strain specific analysis of SERPINA1 gene expression in wild-type C57BL/6JNifdc mice and B-hSERPINA1*E342K mice by RT-qPCR. Liver RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) (female and male, 7-week-old, n=3) and homozygous B-hSERPINA1*E342K mice (H/H) (female and male, 7-week-old, n=3). Values are expressed as mean ± SEM. ND: Not Detectable.

Strain specific analysis of SERPINA1 gene expression in wild-type C57BL/6JNifdc mice and B-hSERPINA1*E342K mice by RT-qPCR. Lung RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) (female and male, 7-week-old, n=3) and homozygous B-hSERPINA1*E342K mice (H/H) (female and male, 7-week-old, n=3). Values are expressed as mean ± SEM. ND: Not Detectable.

The inhibitory efficiency of the nucleic acid drugs against human SERPINA1 in B-hSERPINA1*E342K mice. B-hSERPINA1*E342K mice (female, 8-week-old) were randomly divided into two groups (n=5). The human SERPINA1 targeted nucleic acid drugs, Fazirsiran analog (4 mg/kg, Day 0 and 14, purchased from MCE, HY-132604A) and vehicle (0.9% Saline) were administered to the mice individually and serum samples were collected at the indicated time points (Days -7, 1, 8, 15, 22). (A) Schematic timeline of the in vivo experimental procedure. (B) Human SERPINA1 protein concentrations (μg/mL) measured in serum samples from G1 (vehicle) and G2 (Fazirsiran-analog) groups at each time point. Human SERPINA1 protein was significantly decreased after Fazirsiran analog dosage and B-hSERPINA1*E342K mice can be used in small nucleic acid drug screening research. Significance was determined by t-test, ***P<0.001. Values are expressed as mean ± SEM.