H11-Alb-hTTR*V50M Mice

Figure 1. Gene editing strategy of H11-Alb-hTTR*V50M mice. The "Alb promoter-Human TTR Genomic DNA (exon 1 to ~ 0.5 kb downstream of exon 4)" cassette was inserted into the H11 locus (Approx 0.7 kb upstream of the Eif4enif1 gene and 4.5 kb downstream of the Drg1 gene). The p.V50M (GTG to ATG) point mutation was introduced into human TTR exon 2.

● Research on hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN);

● Preclinical evaluation of TTR-targeted drugs.

1. Detection of human TTR gene and mouse Ttr gene expression

Figure 2. Relative expression levels of human TTR mRNA and mouse Ttr mRNA in the liver and ileum of 3-month-old male homozygous H11-Alb-hTTR*V50M mice, B6-hTTR mice*, and wild-type mice (WT). The RT-qPCR results indicate significant expression of the human TTR gene in the liver and ileum of both H11-Alb-hTTR*V50M mice and B6-hTTR mice, with the H11-Alb-hTTR*V50M mice carrying the pathogenic mutation showing significantly higher expression of human TTR mRNA compared to wild-type humanized B6-hTTR mice. In contrast, wild-type mice (WT) do not express the human TTR gene. Since H11-Alb-hTTRV50M mice retain the mouse Ttr gene, and its expression remains unaffected. Consequently, both wild-type mice (WT) and H11-Alb-hTTR*V50M mice demonstrate mouse Ttr gene expression in their liver and ileum. However, B6-hTTR mice, which lack the mouse Ttr gene, do not exhibit any expression of the mouse Ttr gene.
(ND: Not detected.)
*B6-hTTR mice (Catalog Number: C001512) are a humanized mouse model of the Ttr gene. In this model, the upstream 7.2 kb to 3’ UTR region of the mouse Ttr gene is replaced with the corresponding 7.2 kb to 3’ UTR region of the human TTR gene. For detailed information, please refer to the product manual for this model.

2. ELISA for expression of human TTR protein

Figure 3. ELISA analysis for 3-month-old male homozygous H11-Alb-hTTR*V50M mice, B6-hTTR mice, and wild-type mice (WT). The results indicate that the expression of human TTR protein in the liver and ileum of H11-Alb-hTTR*V50M mice is significantly higher than in the wild-type humanized B6-hTTR mice. In contrast, wild-type mice (WT) do not express human TTR protein.

1. Basic information about the TTR gene

https://rddc.tsinghua-gd.org/en/gene/7276

Transthyretin Amyloidosis (ATTR) is a protein disorder caused by abnormal accumulation of misfolded transthyretin (TTR) protein in various organs and tissues throughout the body. ATTR can be classified into two types: Hereditary/Mutant Type (ATTRv) and Non-hereditary/Wild-Type (ATTRwt). ATTRv is Caused by genetic mutations in the TTR gene. Mutations in the TTR gene lead to the accumulation of abnormal amyloid proteins, which can damage organs and tissues, including the peripheral nervous system and the heart. This condition can result in challenging-to-treat peripheral sensory-motor neuropathy, autonomic neuropathy, and cardiomyopathy ^[1].

4. TTR gene and mutations

The TTR gene encodes transthyretin (TTR), also known as prealbumin. This protein is primarily synthesized in the liver, with a smaller portion formed in the choroid plexus of the brain or the light-sensitive tissue in the eye (such as the retina). TTR functions as a transport protein and, under normal physiological conditions, exists as a homotetramer in peripheral blood. It plays a crucial role in transporting both thyroxine (a thyroid hormone) and retinol-binding protein. Mutations in the TTR gene can lead to hereditary familial amyloidosis, specifically: Transthyretin Cardiac Amyloidosis Myocardiopathy (ATTR-CM) and Transthyretin Amyloid Polyneuropathy (ATTR-PN). Abnormal TTR protein tetramers, due to structural instability, accumulate pathologically in tissues such as the peripheral nervous system, heart, eyes, kidneys, and meninges. This results in insoluble amyloid deposits and ultimately triggers ATTR. ATTR is an autosomal dominant inherited disease, and over 130 mutations have been identified as causative factors. The most common mutations are Val30Met, namely V50M, followed by Val122lIle ^[3]. In addition, the Val50Met (V50M) mutation, also known as c.148G>A and V30M, is the most common mutation in patients with hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) ^[8]. Most ATTRv patients with the Val50Met mutation, if left untreated, will progress to complete disability or even death within 10 to 15 years after diagnosis. Their neurological symptoms may also be accompanied by gastrointestinal dysfunction, cardiomyopathy, renal disease, or ocular deposits.

5. TTR-targeted gene therapy

The treatments for ATTR mainly involve inhibiting the production of mutant TTR gene mRNA or stabilizing the structure of TTR protein tetramers. At present, various drug pipelines have emerged in the field of gene therapy targeting TTR, including ASO, siRNA, and CRISPR-based gene therapies, all of which work by reducing the expression of TTR protein and reducing the accumulation of pathogenic protein. Intellia’s pipeline NTLA-2001 delivers a CRISPR gene editing system targeting the TTR gene to the human body to specifically knock out the TTR gene in liver cells ^[4]. Inotersen sodium, developed by leading gene therapy company Ionis, which is the first approved ASO drug for this disease worldwide. This drug targets the conserved sequence of the 3’untranslated region of TTR mRNA to induce mRNA degradation and reduce TTR synthesis in liver cells. The disease model used in its preclinical research was transgenic TTR (Ile84Ser) mice ^[2]. In addition, preclinical studies of siRNA drugs used transgenic TTR (V30M) mice ^[5-6].

6. Summary

The TTR gene is an important pathogenic gene for Transthyretin amyloidosis (ATTR). Cyagen has developed TTR full-genome humanized mice (B6-hTTR mice, catalog number: C001512) as well as hot mutation disease models (H11-Alb-hTTR*V50M mice, catalog number: C001525). These models can be used for preclinical research in ATTR gene therapy. Additionally, Cyagen offers customized services to meet the experimental needs.