Developing splice-switching oligonucleotides for urea cycle disorder using an integrated diagnostic and therapeutic...

Area of Science:

• Genetics
• Molecular Biology
• Therapeutics

Background:

• Citrin deficiency (CD) is an autosomal recessive urea cycle disorder caused by SLC25A13 gene variants.
• Deep intronic variants often cause genetic diseases and are missed by standard diagnostic tools.
• Identifying and treating these variants is crucial for patients with CD.

Purpose of the Study:

• To develop a workflow for diagnosing and treating splice-altering deep intronic variants in CD.
• To identify novel variants in the SLC25A13 gene causing CD.
• To design and validate splice-switching oligonucleotide (SSO) therapy for CD.

Main Methods:

• A deep intronic-gene panel and RNA analysis were used to identify variants.
• Splice-switching oligonucleotides (SSOs) were designed and validated in vitro using minigene assays and induced hepatocytes.
• In vivo efficacy was assessed in a mouse model.

Main Results:

• A novel SLC25A13 deep intronic variant (c.469-2922G>T) was identified, causing CD by promoting a pseudo-exon.
• Potent SSOs were developed, inhibiting the pseudo-exon with EC50 <2 nM.
• GalNAc-conjugated SSOs restored normal protein expression and urea cycle functions in patient-derived hepatocytes and showed in vivo efficacy without toxicity.

Conclusions:

• A platform was validated for redefining molecular diagnosis of urea cycle disorders.
• Proof-of-concept for precision therapy using SSOs in CD was established.
• This integrated approach can be extrapolated to other rare genetic diseases.