B6-hATXN3 Mice

Figure 1. Gene editing strategy of B6-hATXN3 mice. The sequences from the ATG start codon to ~500bp downstream of exon 11 of the mouse Atxn3 gene were replaced with the sequences from the ATG start codon to ~1 kb downstream of exon 11 of the human ATXN3 gene.

1. Detection of human ATXN3 gene and mouse Atxn3 gene expression

Figure 2. Expression of human ATXN3 gene and mouse Atxn3 gene in the brain and lungs of 6-week-old female wild-type mice (WT), B6-hATXN3 mice, and B6-TG(ATXN3-84Q) mice*. RT-qPCR analysis reveals that the brain and lungs of B6-hATXN3 mice exclusively express the human ATXN3 gene, while the brain and lungs of wild-type mice exclusively express the mouse Atxn3 gene. In contrast, the brain and lungs of B6-TG(ATXN3-84Q) mice express both the human ATXN3 gene and the mouse Atxn3 gene.
*B6-TG(ATXN3-84Q) mouse (catalog number: C001434) is a transgenic model constructed by overexpressing human ATXN3 gene carrying 84 CAG repeat expansions (Q), which is related to the severe form of SCA3. Due to the random insertion of the human ATXN3-84Q gene expression element caused by transgenic technology, this model retains the expression of endogenous genes and proteins in mice while expressing pathogenic human ATXN3 genes and proteins. For more information, please refer to the product manual.
ND: Not detected.

2. Western Blot analysis of ATXN3 protein expression

Figure 3. Expression of ATXN3 protein in the brains of 6-week-old female wild-type mice, B6-hATXN3 mice, and B6-TG(ATXN3-84Q) mice*. Western blot analysis showed that the brains of wild-type mice expressed only murine ATXN3 protein (approximately 42 kDa), while the brains of B6-hATXN3 mice expressed only human ATXN3 protein (approximately 48 kDa)**. The brains of B6-TG(ATXN3-84Q) mice expressed both long polyglutamine-expanded human ATXN3 protein and murine ATXN3 protein, with the observed bands of human ATXN3-84Q protein (approximately 65 kDa) and murine ATXN3 protein (approximately 42 kDa) consistent with the expected results.
*The antibody used in this detection is the recombinant Anti-Ataxin 3 antibody [EPR24153-2] (Abcam, ab259828).
**The human ATXN3 gene and the murine Atxn3 gene are highly homologous. The human ATXN3 protein (361aa) has a slightly longer amino acid sequence than the murine ATXN3 protein (355aa). Although the predicted molecular weights of both the endogenous murine ATXN3 protein and the human ATXN3 protein are approximately 42 KD, the murine Atxn3 gene contains few or no CAG repeats, while the number of CAG repeats in the human ATXN3 gene in healthy individuals ranges from 12 to 44. Consequently, the longer polyQ structure in the human ATXN3 protein may slow down its electrophoretic migration speed in Western Blot detection, resulting in an observed band size larger than the predicted value ^[4].

1. Basic information about the ATXN3 gene

https://rddc.tsinghua-gd.org/gene/4287

2. ATXN3 clinical variants

https://rddc.tsinghua-gd.org/ai/pathogenicity/121381a0f20c4c858ab218cf9f7b2f1b

3. Disease introduction

Spinocerebellar ataxia (SCAs) is a group of genetic diseases characterized by chronic progressive ataxia, which is often inherited in an autosomal dominant pattern but can also be inherited in an autosomal recessive or X-linked pattern. The incidence of SCA ranges from 0 to 5.6 cases per 100,000 people, with an average of 2.7 cases per 100,000 people [1]. The main lesion site is the cerebellum and its associated tissues, which share the common features of movement disorders, including gait ataxia, sudden falls, and speech difficulties. According to the type of gene mutation, SCA can be divided into two categories: repeat expansion SCA and non-repeat expansion SCA. Repeat expansion SCA includes polyglutamine SCA and non-coding region repeat expansion SCA. Among them, SCA3 belongs to the polyglutamine repeat expansion SCA and is the most common type of SCA worldwide, accounting for about 20-50% of cases, followed by SCA2 (13-18%) and SCA6 (13-15%). SCA3 is also known as Machado-Joseph disease (MJD), which is inherited in an autosomal dominant pattern and manifests as cerebellar ataxia, ophthalmoplegia, gaze-evoked nystagmus, eyelid retraction (protrusion sign), facial and lingual fasciculations, varying degrees of pyramidal and extrapyramidal symptoms, and peripheral neuropathy. In addition, SCA has multiple pathogenic mechanisms involving gene dynamic mutation, protein toxicity, RNA toxicity, ion channel dysfunction, etc ^[2-4].

4. ATXN3 gene and mutations

The ATXN3 gene is located on chromosome 14q32.1 and encodes the Ataxin-3 protein, which is widely expressed in the cytoplasm and nucleus of the central nervous system and other tissues. The (CAG)n repeat sequence in exon 10 of the ATXN3 gene can undergo abnormal amplification mutation, leading to abnormal aggregation of polyglutamine in nerve tissue and eventually causing SCA. In general, the severity of the disease is positively correlated with the number of CAG repeats. The severity of the disease increases with an increase in CAG repeats, and the onset occurs earlier. The normal range of CAG repeats in the ATXN3 gene is 12-44, while the range of CAG repeats in SCA3 patients increases to 56-87 ^[2-4].

5. Function of non-coding DNA sequences

Mutations in the 3’UTR region of the ATXN3 gene can affect the age of onset of SCA3 (MJD) patients [6]. miR-25 and miR-181 can cooperatively bind to the 3’UTR to inhibit abnormal expression of ATXN3, which may be a potential drug target for treating SCA3 (MJD) ^[7].

6. ATXN3-targeted gene therapy

Currently, most of the therapies targeting ATXN3 for SCA are still in the early stages of development. The treatment strategy is to target ATXN3 with miRNA or ASO drugs to reduce its abnormal expression. A joint research result from the University of Michigan and Ionis showed that ASO drugs can inhibit the expression of mutant ATXN3 in SCA3 transgenic mice (Q84/Q84) and rescue their motor impairment ^[8]. A preclinical study published by uniQure showed that injecting AAV5-miATXN3 into a disease model can effectively inhibit the abnormal expression of ATXN3, and the disease model used has 304 glutamine repeat sequences ^[9].

In summary, the ATXN3 gene is an important pathogenic gene for Machado-Joseph disease (MJD, i.e., SCA3), and its pathogenic mechanism is complex. ATXN3 whole-genome humanized mice from Cyagen can be used for preclinical research on SCA3, and customized services can also be provided for different point mutations.