Spinocerebellar Ataxia (SCA) is a group of genetically inherited (autosomal dominant) brain diseases characterized by progressive and degenerative features. These diseases primarily affect the cerebellum's regulation of body movement coordination, and sometimes also impact the spinal cord. Over 40 subtypes of SCA have been identified, each associated with a specific pathogenic, or disease-causing, gene.[1] The main symptom of SCA is a gradual decline in gait coordination, usually accompanied by coordination problems with the hands, as well as speech and eye movements. Specific symptoms vary depending on the subtype and individual differences among patients. In many cases, despite patients' cognitive abilities remaining intact, they suffer from a gradual loss of motor control, which can lead to the loss of mobility and even death.
Figure 1: Global Main Distribution of Spinocerebellar Ataxia (SCA) [1]
Spinocerebellar Ataxia (SCA) is primarily caused by the expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in genes, characteristic of the polyglutamine (polyQ) disease group. Most cases of SCA, such as SCA1, SCA2, and SCA3, belong to this category. The mechanism of these diseases involves the increase in the length of the pathogenic polyglutamine expansion repeat sequences, leading to the production of abnormal PolyQ repeat sequences during translation, which causes protein misfolding. The misfolded PolyQ proteins form aggregates, disrupting cellular processes and leading to cellular dysfunction, ultimately causing cytotoxicity and degeneration. Other genetic alterations such as point mutations, deletions, or insertions at different gene loci are relatively rare. Spinocerebellar Ataxia Type 3 (SCA3), also known as Machado-Joseph Disease (MJD), is the most common and severe subtype of SCA globally, accounting for about 20% to 50% of all SCA cases, although a cure has yet to be discovered.[2]
Figure 2: The function of normal CAG trinucleotide repeats and the mechanism by which abnormal expansion of CAG trinucleotide repeats leads to the onset of SCA diseases [2]
In human bodies, the number of CAG repeats in the ATXN3 gene normally ranges from 10 to 44. However, in patients with SCA3, this range extends from 61 to 87. This excessive CAG repetition leads to the mutated ATXN3 gene encoding the production of abnormal ataxin-3 proteins, which cannot function properly. This is especially impactful since the ataxin-3 protein is involved in a mechanism called the ubiquitin-proteasome system that destroys and gets rid of excess or damaged proteins. As these abnormal ataxin-3 proteins aggregate, toxic substances begin building-up, disrupting multiple cellular processes including autophagy, protein homeostasis, transcription, mitochondrial function, and signal transduction – resulting in functional impairments and indicating onset of the disease. Symptoms in patients include cerebellar ataxia, paralysis of extraocular muscles, gaze-evoked nystagmus (GEN), eyelid retraction, dysphagia, facial and tongue muscle twitching, as well as varying degrees of pyramidal and extrapyramidal symptoms, including peripheral neuropathy.[3]
Figure 3: The impact of varying CAG repeat numbers in the ATXN3 gene on the function of Ataxin-3 protein.[3]
In the current preclinical research landscape, certain therapeutic effects have been demonstrated through several approaches, including: inhibiting the expression of the ATXN3 gene, silencing the pathogenic ATXN3 protein, preventing protein aggregation, inhibiting the degradation of toxic proteins, and counteracting the dysfunction of affected cellular systems.[3] However, there are a very limited number of SCA3 therapies that have reached the clinical evaluation stage, indicating further research and modeling tools are needed. Depending on the therapeutic approach, each treatment must be rigorously assessed using different types of animal models before proceeding to clinical trials. For example, Ionis Pharmaceuticals, a leading company specializing in small nucleic acid drugs, has used a variety of mouse models expressing the human ATXN3 gene for target molecule screening and pharmacological validation in the development of ATXN3-targeted antisense oligonucleotide (ASO) therapies.[4-7]
Figure 4: Targeting ATXN3 mRNA or ATXN3 protein and various other SCA3 therapeutic approaches.[3]
Cyagen has successfully developed the humanized B6-hATXN3 mouse model with the humanized Atxn3 gene (Product Number: C001398). This model can be further customized to incorporate hot-spot pathogenic mutations to meet the research needs for emerging therapies such as Targeted Gene Editing, antisense oligonucleotides (ASO), small interfering RNA (siRNA), and microRNA (miRNA). Here are the detailed specifications of this model.
The common B6-TG(ATXN3-84Q) mice express both the human ATXN3 gene and the mouse Atxn3 gene. RT-qPCR results show that the expression levels of the human ATXN3 gene in B6-hATXN3 mice are close to those in B6-TG(ATXN3-84Q) mice. Moreover, B6-hATXN3 mice exclusively express the human gene and do not express the mouse gene.
Figure 5: Detection of gene expression in wild-type mice (B6N), B6-hATXN3 mice, and B6-TG(ATXN3-84Q) mice
Western Blot results show that the brains of wild-type mice only express the mouse ATXN3 protein (approximately 42 kDa), while the brains of B6-hATXN3 mice exclusively express the human ATXN3 protein (approximately 48 kDa). The brains of the B6-TG(ATXN3-84Q) mouse model express both the human ATXN3 protein with an elongated PolyQ structure mutation and the mouse ATXN3 protein, showing bands for human ATXN3-84Q protein (approximately 65 kDa) and mouse ATXN3 protein (approximately 42 kDa).
Figure 6: Western Blot (WB) Detection of human ATXN3 protein expression in mouse brain tissue.
The B6-hATXN3 mouse model (Product Number: C001398) effectively expresses the human ATXN3 gene and does not express the mouse endogenous Atxn3 gene. There is significant expression of human ATXN3 protein in its brain. The commonly-used B6-TG(ATXN3-84Q) mouse model, which expresses both the human ATXN3 gene and the mouse Atxn3 gene, may provide difficulties in efficient translation from research discovery through achieving effective preclinical and clinical results. Therefore, the B6-hATXN3 mouse model can be used for the study of Spinocerebellar Ataxia Type 3 (SCA3) disease and discovering potential therapeutics.
Additionally, Cyagen utilizes its proprietary TurboKnockout fusion BAC recombination technology to offer point mutation disease models, customized to provide patient-specific and/or hotspots of human mutations based on this wild-type humanization model, providing customized services to meet the needs of SCA3 disease researchers for drug screening and pharmacological efficacy experiments.
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References:
[1] Klockgether T, Mariotti C, Paulson HL. Spinocerebellar ataxia. Nat Rev Dis Primers. 2019 Apr 11;5(1):24.
[2] Sullivan R, Yau WY, O'Connor E, Houlden H. Spinocerebellar ataxia: an update. J Neurol. 2019 Feb;266(2):533-544. doi: 10.1007/s00415-018-9076-4. Epub 2018 Oct 3.
[3] Matos CA, de Almeida LP, Nóbrega C. Machado-Joseph disease/spinocerebellar ataxia type 3: lessons from disease pathogenesis and clues into therapy. J Neurochem. 2019 Jan;148(1):8-28.
[4] Moore LR, Rajpal G, Dillingham IT, Qutob M, Blumenstein KG, Gattis D, Hung G, Kordasiewicz HB, Paulson HL, McLoughlin HS. Evaluation of Antisense Oligonucleotides Targeting ATXN3 in SCA3 Mouse Models. Mol Ther Nucleic Acids. 2017 Jun 16;7:200-210.
[5] Toonen LJA, Rigo F, van Attikum H, van Roon-Mom WMC. Antisense Oligonucleotide-Mediated Removal of the Polyglutamine Repeat in Spinocerebellar Ataxia Type 3 Mice. Mol Ther Nucleic Acids. 2017 Sep 15;8:232-242.
[6] McLoughlin HS, Moore LR, Chopra R, Komlo R, McKenzie M, Blumenstein KG, Zhao H, Kordasiewicz HB, Shakkottai VG, Paulson HL. Oligonucleotide therapy mitigates disease in spinocerebellar ataxia type 3 mice. Ann Neurol. 2018 Jul;84(1):64-77.
[7] McLoughlin HS, Gundry K, Rainwater O, Schuster KH, Wellik IG, Zalon AJ, Benneyworth MA, Eberly LE, Öz G. Antisense Oligonucleotide Silencing Reverses Abnormal Neurochemistry in Spinocerebellar Ataxia 3 Mice. Ann Neurol. 2023 Oct;94(4):658-671.
