hTTR-V30M Mouse Model

The humanized hTTR-V30M mouse model was successfully established by precisely inserting the human TTR sequence (containing the V30M mutation) after the start codon ATG and before the stop codon TGA of the mouse Ttr gene in C57BL/6N mice. In this model, human TTR (with the V30M mutation) is successfully expressed, while the expression of the murine Ttr is terminated.

Hereditary transthyretin amyloidosis (ATTRv) is an autosomal dominant systemic amyloidosis caused by pathogenic mutations in the TTR gene. V30M (Val30Met; p.Val50Met according to HGVS nomenclature including the signal peptide) is one of the most representative and well-studied neuropathy-associated variants. ATTRv can manifest as progressive sensory and autonomic peripheral neuropathy, autonomic dysfunction, gastrointestinal symptoms, and nutritional decline, and may be accompanied by myocardial involvement. As the disease progresses, patients may experience severe functional impairment, decreased quality of life, and death. In recent years, the treatment of ATTRv has progressively expanded from liver transplantation and supportive care to disease-modifying therapeutic strategies, such as TTR tetramer stabilizers, RNA interference therapeutics, antisense oligonucleotides, and in vivo gene editing. Because different treatment modalities act on different pathological levels—such as tetramer stabilization, hepatic TTR synthesis inhibition, or TTR gene inactivation—there is still a need for a stable, reproducible preclinical in vivo evaluation system capable of reflecting human TTR-V30M expression, conformational changes, tissue deposition, and pharmacodynamic endpoints.

TTR is primarily synthesized by the liver and can also be produced by the choroid plexus and retinal pigment epithelium. Its physiological functions include serving as a minor transport protein for thyroxine and forming a complex with retinol-binding protein 4 (RBP4) to participate in vitamin A transport. Circulating TTR mainly exists in the form of a homotetramer. Tetramer dissociation, monomer misfolding, and subsequent oligomer and amyloid fibril formation are considered key pathological processes in TTR amyloidosis; pathogenic TTR mutations typically promote amyloidogenesis by reducing tetramer stability and/or accelerating tetramer dissociation. Early studies established transgenic mouse models by introducing the human mutant TTR Met30 gene, revealing that amyloid deposition can appear in the gastrointestinal tract, cardiovascular system, and kidneys starting from approximately 6 months of age, and extend to other tissues with increasing age. By 24 months of age, the distribution of deposition in this model showed certain similarities to autopsy cases of human familial amyloidotic polyneuropathy, but corresponding depositions were absent in the choroid plexus, peripheral nerves, and autonomic nervous system. Therefore, this model is suitable for studying the initiation and progression of TTR amyloid deposition, but cannot be regarded as a humanized model that fully recapitulates the neuropathological phenotype of ATTRv.

Drug development surrounding TTR-V30M/ATTRv has formed a relatively clear mechanistic stratification. Tafamidis kinetically stabilizes the TTR tetramer by binding to its thyroxine-binding sites, thereby slowing monomer formation, misfolding, and amyloidogenesis, and is a representative tetramer stabilizer for early Val30Met-related diseases. Patisiran is a small interfering RNA (siRNA) therapeutic that inhibits hepatic TTR synthesis via RNA interference, reduces circulating mutant and wild-type TTR levels, and has demonstrated disease-modifying effects in patients with ATTRv polyneuropathy. Inotersen is a 2′-O-methoxyethyl-modified antisense oligonucleotide (ASO) that similarly reduces mutant and wild-type TTR levels by inhibiting hepatic TTR production, and has completed randomized, double-blind, placebo-controlled clinical studies. In addition, NTLA-2001 utilizes lipid nanoparticles to deliver Cas9 mRNA and a TTR-targeted single guide RNA, achieving TTR gene inactivation via in vivo CRISPR-Cas9 gene editing, representing a directional expansion of TTR-targeted therapy from protein stabilization and RNA reduction further to gene editing.

These therapeutic strategies further highlight the necessity of humanized models. Establishing the hTTR-V30M humanized mouse model can facilitate the evaluation of TTR tetramer stabilizers, siRNAs, ASOs, gene-editing therapeutics, and other candidate therapies targeting human TTR or amyloid deposition. This model should focus on supporting the evaluation of human TTR expression and serum exposure, tetramer stability, tissue deposition, pharmacodynamics, dose optimization, combination therapy, pathological progression, and toxicity-related endpoints, thereby providing a stable and reproducible in vivo platform for mechanistic research and preclinical translation of ATTRv/TTR-V30M-targeted drugs.

[1] Adams D, Koike H, Slama M, Coelho T. Hereditary transthyretin amyloidosis: a model of medical progress for a fatal disease. Nat Rev Neurol. 2019;15(7):387-404.

[2] Coelho T, Merlini G, Bulawa CE, Fleming JA, Judge DP, Kelly JW, et al. Mechanism of Action and Clinical Application of Tafamidis in Hereditary Transthyretin Amyloidosis. Neurol Ther. 2016;5(1):1-25.

[3] Yi S, Takahashi K, Naito M, Tashiro F, Wakasugi S, Maeda S, et al. Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met30) gene. Pathologic similarity to human familial amyloidotic polyneuropathy, type I. Am J Pathol. 1991;138(2):403-12.

[4] Adams D, Gonzalez-Duarte A, O’Riordan WD, Yang CC, Ueda M, Kristen AV, et al. Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. N Engl J Med. 2018;379(1):11-21.

[5] Benson MD, Waddington-Cruz M, Berk JL, Polydefkis M, Dyck PJ, Wang AK, et al. Inotersen Treatment for Patients with Hereditary Transthyretin Amyloidosis. N Engl J Med. 2018;379(1):22-31.

[6] Gillmore JD, Gane E, Taubel J, Kao J, Fontana M, Maitland ML, et al. CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. N Engl J Med. 2021;385(6):493-502.