Antisense Oligonucleotide Enhances SLC20A2 Expression and Inhibits Brain Calcification in Humanized Mouse Models

Background and Research Questions

Primary Familial Brain Calcification (PFBC) is an age-related neurogenetic disorder, characterized by bilateral calcifications in brain regions such as the basal ganglia, thalamus, and cerebellum. PFBC patients often exhibit a range of symptoms including headache, Parkinsonian movement disorders, cognitive decline, anxiety, and depression. Currently, clinical management of PFBC relies solely on symptomatic treatment, with no therapies available to effectively inhibit the progression of brain calcification.

The genetic basis of PFBC includes mutations in genes such as SLC20A2, PDGFRB, and PDGFB, with approximately 61% of PFBC cases attributed to heterozygous mutations in the SLC20A2 gene. However, the pathological mechanism by which SLC20A2 mutations lead to brain calcification remains unclear. The authors of this paper have identified a novel deep intronic mutation in the SLC20A2 gene and explored the possibility of restoring gene expression through antisense oligonucleotides (ASOs) to regulate mRNA splicing.

Source and Authors

This study was conducted by Miao Zhao et al., with main contributors from Fujian Medical University, the Shanghai Brain Science and Brain-like Technology Center, and the Chinese Academy of Sciences Institute of Neuroscience. The research was published in Neuron on October 9, 2024.

Research Design and Process

Samples and Genetic Analysis

The research team conducted whole-genome sequencing (WGS) on 135 PFBC patients with no abnormalities found through exon sequencing, identifying 5 types of deep intronic mutations in the SLC20A2 gene across 6 families. These mutations were validated through Sanger sequencing, and computational prediction analyses suggested that they could affect mRNA splicing sites.

Molecular Mechanism Study

Utilizing minigene splicing models, the team explored how these intronic mutations lead to aberrant splicing. The results demonstrated that the mutations introduced novel cryptic exons, which increased the production of non-functional mRNA by affecting mRNA splicing, resulting in insufficient SLC20A2 protein expression.

Additionally, through RNA pull-down and mass spectrometry analyses, the team confirmed changes in the binding affinity of specific RNA-binding proteins (such as SRSF3 and hnRNPQ) with the cryptic exons, further explaining how the mutations interfere with splicing regulatory mechanisms.

Cellular Experiments and ASO Intervention

The team designed ASOs targeting the cryptic exons to inhibit abnormal splicing by stably binding to RNA and preventing spliceosome access to related sites. Cell experiments demonstrated that ASOs significantly reduced cryptic exon-mediated abnormal mRNA production and restored SLC20A2 protein expression levels. This result was validated in fibroblasts derived from PFBC patients.

Verification in Humanized Mouse Models

Researchers created a humanized knock-in mouse model carrying the human SLC20A2 mutated intronic sequences to mimic the brain calcification pathology observed in PFBC patients. Compared to wild-type mice, the knock-in mice displayed elevated levels of inorganic phosphate (Pi) in cerebrospinal fluid and progressive brain calcification.

Through intracerebroventricular (ICV) injection of ASOs, the researchers successfully reduced cerebrospinal fluid Pi levels and significantly inhibited brain calcification progression in knock-in mice, confirming the effectiveness of ASO therapy.

Main Findings and Results

1. Genetic Discovery: For the first time, established that 5 types of deep intronic mutations led to cryptic exon-mediated aberrant splicing of the SLC20A2 gene, resulting in reduced protein expression.
2. Cellular Validation: ASOs effectively restored SLC20A2 protein expression in patient-derived cells by inhibiting cryptic exon splicing.
3. Mouse Model Validation: In humanized mice, ASO injection reduced Pi levels in cerebrospinal fluid and slowed brain calcification progression.
4. Therapeutic Potential: ASO-mediated splicing correction provides a feasible treatment strategy for PFBC caused by SLC20A2 insufficiency.

Research Significance and Value

Scientific Value

This study systematically elucidated the molecular mechanisms by which deep intronic mutations cause gene function loss through cryptic exons, extending the genetic etiology of PFBC. The research also provided a new mouse model for diseases related to the SLC20A2 gene, facilitating in-depth exploration of its pathological mechanisms.

Clinical Value

The successful application of ASO therapy in other genetic diseases (such as spinal muscular atrophy and Duchenne muscular dystrophy) suggests that ASO-mediated splicing regulation holds clinical translational potential. The “CRACE” strategy (Calcification Repression by ASO Corrected Expression) proposed herein not only effectively slows PFBC’s calcification progression but also offers therapeutic insights for other diseases associated with Pi homeostasis disorders (e.g., chronic kidney disease and cardiovascular disease).

Highlights and Innovations

1. First verification of the pathogenic mechanism of deep intronic mutations in SLC20A2.
2. Proposed an ASO-based “CRACE” therapeutic strategy, providing a new direction for PFBC and other genetic diseases.
3. Developed an efficient knock-in mouse model simulating PFBC, facilitating further research and drug screening.

Prospects and Future Directions

Future research should further optimize molecular modifications and delivery methods of ASOs to enhance the regional specificity and dose efficiency of the therapy. Meanwhile, combining multiple ASOs for treatment might enhance efficacy and lay the foundation for precision medicine.

The important findings and technological innovations in this study demonstrate the potential of gene splicing regulation in treating genetic diseases, providing a solid basis for developing novel therapies.