Compositions and methods for treatment of white matter dystrophy and intact animal and cell models for identification of effective drugs for treatment of white matter dystrophy
A technology for dystrophy and leukodystrophy, applied in the field of compositions for treating leukodystrophy and complete animal and cell models for identifying effective drugs for treating leukodystrophy, and can solve problems such as no animal model
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Embodiment I
[0182] A Mouse Model of H-ABC Disease
[0183] In this example we will generate a Tubb4a D249N / D249N Mutant knock-in mice serve as an H-ABC model that recapitulates features of the human disease, including dystonia, loss of motor function, and gait abnormalities. Histopathological features of the mouse model include neuronal loss in the striatum and cerebellum and hypomyelination in the brain and spinal cord observed in patient tissue (Curiel et al., 2017b). We also use Tubb4a D249N / D249N Functional consequences of mutant tubulin on microtubule polymerization and cell-autonomous effects of Tubb4a mutations were explored in neurons and oligodendrocytes in mice. This study provides a promising first model for H-ABC using the most common mutations, which is key to understanding the mechanisms underlying this devastating disease and developing therapies.
[0184] Tubb4a D249N Generation of CRISPR knock-in mice
[0185] To understand the molecular mechanisms and disease progr...
Embodiment II
[0289] TUBB4A Human iPS Cells
[0290] As discussed in the previous example, hypomyelination of the basal ganglia and cerebellum (H-ABC) is a rare leukodystrophy that our group has determined to be sporadic in the TUBB4A gene. Caused by a de novo heterozygous mutation (Simons et al. 2013). Monoallelic mutations in TUBB4A may cause a range of neurological disorders, from early-onset encephalopathy to adult dystonia type 4 (whispering dysphonia). Individuals affected by H-ABC fall within this spectrum, presenting in early childhood, often with dystonia (Hersheson et al. 2013), progressive gait disturbance, and speech and cognitive deficits. They are further distinguished from other individuals with TUBB4A mutations by the following characteristic neuroimaging features: hypomyelination and atrophy of the caudate nucleus and putamen and cerebellar atrophy (van der Knaap et al. 2007). In pathological specimens, the dorsal striatal region and cerebellar granular layer showed neuro...
Embodiment III
[0295] Antisense molecules for down-regulation of the target TUBB4A gene
[0296]The inventors have developed a series of antisense oligonucleotides that are effective in downregulating the overall level of TUBB4A gene expression. The methods described below are capable of downregulating wild-type and mutant TUBB4-A expression in target cells of interest.
[0297] Eleven ASOs were synthesized by Integrated DNA Technologies. We performed an in vitro screen of these ASOs to identify the optimal ASO design. Mouse Oli-neu cells were electroporated with ASO concentrations of 1 μM, 5 μM and 10 μM in 100 μL medium with 100,000 cells / well at a voltage of 150 V on a NEPA21 electroporation system (NEPA GENE, USA). After electroporation, cells were transferred to poly-L-ornithine-coated plates and placed in an incubator. Forty-eight hours after treatment, cells were washed with PBS before using PureLink TM RNA Mini Kit (ThermoFisher Scientific, Cat: 12183018A) was used for RNA extra...
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