B6-hTTR Mice

Figure 1. Gene editing strategy of B6-hTTR mice. The sequences from ~7.2 kb upstream of exon 1 to exon 4 of the mouse Ttr gene will be replaced with the sequences from ~7.2 kb upstream of exon 1 to exon 4 of the human TTR gene.

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

Figure 2. Human TTR gene and mouse Ttr gene expression in the liver of 6-week-old male homozygous B6-hTTR mice (hTTR) and wild-type mice (B6N). RT-qPCR analysis showed that there was significant expression of human TTR gene in the liver of B6-hTTR mice, while there was no expression of human TTR gene in B6N mice; there was significant expression of mouse Ttr gene in the liver of B6N mice, while there was no expression of mouse Ttr gene in B6-hTTR mice.
ND: Not detected

2. ELISA for human TTR expression

Figure 3. ELISA* detection results of homozygous B6-hTTR mice (hTTR) and wild-type mice (WT) at 6 weeks of age. Human TTR was significantly expressed in the B6-hTTR mice, but not in WT mice. There was no significant difference in human TTR expression between male and female B6-hTTR mice.
*The reagent used in this experiment is the human Prealbumin ELISA kit (Abcam, ab231920).

3. Small interfering RNA (siRNA) drugs significantly reduce the level of TTR in B6-hTTR mice*

Figure 4. ELISA results of TTR expression in the plasma of 4-month-old homozygous male B6-hTTR mice after injection of Vutrisiran**. The results showed that the expression level of human TTR protein in the plasma of B6-hTTR mice was significantly lower than that in the control group treated with NaCl after a single subcutaneous injection of Vutrisiran.
*Vutrisiran (MedChemExpress, HY-132589), also known as ALN-TTRsc02, is a liver-directed small interfering RNA (siRNA) drug that inhibits TTR mRNA via the RNA interference (RNAi) mechanism, thereby reducing serum TTR protein and TTR protein deposition in tissues to achieve therapeutic effects ^[7].
**The assay used was the Human PreAlbumin ELISA kit (Transthyretin) (Abcam, ab108895).

1. Basic information about the TTR gene

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

2. TTR clinical variants

https://rddc.tsinghua-gd.org/en/ai/pathogenicity/result?id=8ee6064e-6138-4f62-bac7-b546edd688ea

3. Disease introduction

Transthyretin amyloidosis (ATTR) is a protein disorder caused by the abnormal accumulation of misfolded transthyretin (TTR) protein in organs and tissues throughout the body. ATTR can be divided into hereditary ATTR and wild-type ATTR, with hereditary ATTR being caused by genetic mutations in the TTR gene. Mutations in the TTR gene can lead to abnormal amyloid protein accumulation, damaging organs and tissues, including the peripheral nervous system and heart, resulting in peripheral sensory-motor neuropathy, autonomic neuropathy, cardiomyopathy, and other conditions [1].

4. TTR gene and mutations

The TTR gene encodes transthyretin (TTR) protein, which is mainly synthesized in the liver and to a lesser extent in the brain’s choroid plexus or ocular photoreceptor tissue (such as the retina). TTR is a transport protein that exists as a homotetramer in peripheral blood under normal physiological conditions and participates in the transport of thyroxine and retinol-binding protein. Mutations in the TTR gene can produce structurally unstable TTR protein tetramers, which pathologically aggregate in tissues such as the peripheral nervous system, heart, eyes, kidneys, and meninges, forming insoluble amyloid deposits, eventually leading to 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]. 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). Cyagen can also provide a popular point-mutated disease model, the H11-Alb-hTTR*V50M mouse (catalog number: C001525), to support preclinical research for ATTR gene therapy. 

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. 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 for Transthyretin amyloidosis (ATTR). As an important target for the treatment of ATTR, various drug pipelines have emerged in the field of gene therapy, including ASO, siRNA, and CRISPR-based gene therapies. TTR gene humanized mice from Cyagen can be used for preclinical research on ATTR gene therapy, and customized services can also be provided for different mutations.