• Background: Transthyretin (TTR) is a tetrameric transport protein primarily synthesized in the liver, choroid plexus, and retinal pigment epithelium. It functions to transport thyroid hormone thyroxine and retinol-binding protein bound to retinol (vitamin A). Mutations in TTR are associated with inherited transthyretin amyloidosis (ATTRv), a progressive and fatal disease characterized by misfolding of TTR and aggregation as amyloid fibrils, predominantly leading to cardiomyopathy or polyneuropathy. Additionally, wild-type TTR misfolding can cause age-related transthyretin amyloid cardiomyopathy. Besides its transport role, TTR may also play a part in neuroprotection and promotion of neurite outgrowth in response to injury.
• Targeting strategy: The exons 1-4 of mouse Ttr gene that encode whole protein domains are replaced by human counterparts containing p.V30M mutation in B-hTTR*V30M mice plus. The promoter, 5’UTR and 3’UTR regions of the mouse gene are replaced by human counterparts, and mouse Ttr gene transcription and translation will be disrupted.
• Validation: Human prealbumin (TTR) was exclusively detectable in B-hTTR*V30M mice plus but not in wild-type mice.
• Application: This is a disease model mouse of transthyretin amyloidotic polyneuropathy (ATTR-PN). It can be used for small nucleic acid drug screening and pharmacodynamic research, as well as for safety evaluation.

Gene targeting strategy for B-hTTR*V30M mice plus. The exons 1-4 of mouse Ttr gene that encode whole protein domains are replaced by human counterparts containing p.V30M mutation in B-hTTR*V30M mice plus. The promoter, 5’UTR and 3’UTR regions of the mouse gene are replaced by human counterparts, and mouse Ttr gene transcription and translation will be disrupted.

Strain specific TTR expression analysis in homozygous B-hTTR*V30M mice plus by ELISA. Plasma was collected from wild-type C57BL/6JNifdc mice (+/+) (female, 8-week-old, n=3) and homozygous B-hTTR*V30M mice plus (H/H) (female, 7-week-old, n=3), and then analyzed by ELISA with species-specific Prealbumin ELISA kit (abcam, ab282297). Mouse prealbumin (TTR) was only detectable in wild-type C57BL/6JNifdc mice but not in homozygous B-hTTR*V30M mice plus.

Strain specific hTTR expression analysis in homozygous B-hTTR*V30M mice plus by ELISA. Plasma was collected from wild-type C57BL/6 mice (+/+) (female, 8-week-old, n=3) and homozygous B-hTTR*V30M mice plus (H/H) (female, 7-week-old, n=3; male, 7-week-old, n=3), and then analyzed by ELISA with species-specific Prealbumin ELISA kit (abcam, ab231920). Human prealbumin (TTR) was exclusively detectable in B-hTTR*V30M mice plus but not in wild-type mice.

The inhibitory efficiency of the nucleic acid drugs against human TTR in B-hTTR*V30M mice plus. B-hTTR*V30M mice plus (male, 8-week-old) were randomly divided into two groups (n=5). The human TTR targeted nucleic acid drugs, Vutrisiran analog (1 mg/kg, Day 0, purchased from MCE, HY-132589) and vehicle (0.9% Saline) were administered to the mice individually and plasma samples were collected at the indicated time points (Days -7, 1, 8, 15, 22). (A) Schematic timeline of the in vivo experimental procedure. (B) Left: Human TTR protein concentrations (μg/mL) measured in plasma samples from G1 (vehicle) and G2 (Vutrisiran-analog) groups at each time point. Right: Residual human TTR protein levels relative to the pre-administration (Day -7) baseline. Human TTR protein was significantly decreased after Vutrisiran analog dosage and B-hTTR*V30M mice plus 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.