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Antisense oligonucleotides enhance SLC20A2 expression and suppress brain calcification in a humanized mouse model
Miao Zhao1, Xuewen Cheng2, Lei Chen3
1Department of Neurology, the First Affiliated Hospital, Institute of Neuroscience, Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou 350005, China; Department of Neurology, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian 350212, China.
Abstract:
Primary familial brain calcification (PFBC) is a genetic neurological disease, yet no effective treatment is currently available. Here, we identified five novel intronic variants in SLC20A2 gene from six PFBC families. Three of these variants increased aberrant SLC20A2 pre-mRNA splicing by altering the binding affinity of splicing machineries to newly characterized cryptic exons, ultimately causing premature termination of SLC20A2 translation. Inhibiting the cryptic-exon incorporation with splice-switching ASOs increased the expression levels of functional SLC20A2 in cells carrying SLC20A2 mutations. Moreover, by knocking in a humanized SLC20A2 intron 2 sequence carrying a PFBC-associated intronic variant, the SLC20A2-KI mice exhibited increased inorganic phosphate (Pi) levels in cerebrospinal fluid (CSF) and progressive brain calcification. Intracerebroventricular administration of ASOs to these SLC20A2-KI mice reduced CSF Pi levels and suppressed brain calcification. Together, our findings expand the genetic etiology of PFBC and demonstrate ASO-mediated splice modulation as a potential therapy for PFBC patients with SLC20A2 haploinsufficiency.
Insights
Researchers identified novel genetic variants in the SLC20A2 gene causing primary familial brain calcification (PFBC). Splice-switching antisense oligonucleotides (ASOs) show promise in restoring functional SLC20A2 and reducing brain calcification in a mouse model.
Area of Science:
- Genetics
- Neuroscience
- Molecular Biology
Background:
- Primary familial brain calcification (PFBC) is a rare genetic neurological disorder.
- Current treatments for PFBC are limited, highlighting the need for novel therapeutic strategies.
Purpose of the Study:
- To identify novel genetic variants associated with PFBC.
- To investigate the molecular mechanisms underlying PFBC caused by intronic variants in SLC20A2.
- To evaluate the therapeutic potential of splice-switching antisense oligonucleotides (ASOs) for PFBC.
Main Methods:
- Genetic analysis of six PFBC families to identify intronic variants in the SLC20A2 gene.
- Assessment of the impact of identified variants on SLC20A2 pre-mRNA splicing and translation.
- Development and testing of splice-switching ASOs to correct aberrant splicing.
- Generation and characterization of a mouse model (SLC20A2-KI) with a PFBC-associated intronic variant.
- Evaluation of ASO-mediated therapeutic effects in the SLC20A2-KI mouse model.
Main Results:
- Five novel intronic variants in SLC20A2 were identified in six PFBC families.
- Three variants were found to disrupt SLC20A2 pre-mRNA splicing, leading to premature translation termination.
- Splice-switching ASOs successfully restored functional SLC20A2 expression in cellular models.
- SLC20A2-KI mice exhibited increased cerebrospinal fluid (CSF) inorganic phosphate (Pi) levels and progressive brain calcification.
- Intracerebroventricular ASO administration in mice reduced CSF Pi levels and suppressed brain calcification.
Conclusions:
- The study expands the known genetic causes of PFBC, implicating novel intronic variants in SLC20A2.
- ASO-mediated splice modulation represents a promising therapeutic approach for PFBC patients with SLC20A2 haploinsufficiency.
- Targeting aberrant splicing offers a potential strategy for treating genetic neurological disorders like PFBC.
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