Like living software, the human genome provides the operational code for the hardware of the human body. Mutations in that code can act like ‘bugs’, disrupting normal biological functions and causing myriad rare diseases. Telling these diseases apart can be critical, as many share near-identical symptoms, yet need very different treatments.
“However, some genetic diseases are caused by deep intronic variants—mutations buried in obscure, non-coding DNA sections—missed by common diagnostic tools,” said Dave Wee and Jin Rong Ow, a Principal Investigator and a Senior Scientist respectively at the A*STAR Institute of Molecular and Cell Biology (A*STAR IMCB).
Wee and Ow noted how Kimihiko Oishi, Eri Imagawa and colleagues at the Jikei University School of Medicine, Japan, brought their team’s attention to this diagnostic gap through a group of patients with citrin deficiency (CD). One of several urea cycle disorders (UCDs), CD disrupts the body’s ability to clear ammonia from the bloodstream. Yet, while patients with CD need high-protein, high-fat diets to maintain stable blood ammonia levels, these diets can be toxic to patients with other UCDs.
“While these patients were clinically diagnosed with CD, standard genetic tests could only find one faulty copy—or none—of the SLC25A13SLC25A13 gene, though the disease is recessive and requires two,” said Ow.
Suspecting a hidden variant in a deep intronic region, the team launched an international, multidisciplinary effort to develop a new gene panel and RNA analytical platform that would uncover what other sequencing tools missed. The effort included A*STAR IMCB Senior Group Leader Manikandan Lakshmanan and Senior Scientist Venkataraman Ramadass, as well as research institutes in Singapore, Japan, Malaysia, the US and Saudi Arabia.
“Our main aim was to provide definitive answers for families by creating an integrated precision medicine platform for rare diseases like CD,” said Wee.
As deep intronic regions contain repetitive DNA sequences that confuse capture probes, creating unreliable results, the team developed a custom-built ‘PRUNE’ algorithm that iteratively filled coverage gaps. By adding high-performing probes and removing off-target ones, PRUNE also ensured higher sequencing coverage and accuracy, and led the team to SLC25A13-PE5, a novel deep intronic variant in CD.
Using their rational splice-switching oligonucleotide (SSO) design pipeline, the team then identified a promising SSO conjugated with GaINAc that could target the liver: the organ central to CD. When tested in patient-derived liver cells, the molecule reversed the effects of SLC25A13-PE5; it also showed no acute toxicity effects in healthy animal models.
“The recovery of citrin function confirms the feasibility of our GaINAc-SSO’s targeted mechanism of action,” said Ow.
Based on their preclinical results, the team’s next steps could include pharmacological tests of their SSO candidate in animal models of CD, clinical-grade production and comprehensive toxicity testing.
“Our workflow is designed to be lean and adaptable, enabling customised ‘N-of-1’ therapies tailored to specific genetic mutations,” said Wee. “We hope this work will inspire confidence that no disease is too rare to be treated.”
The A*STAR-affiliated researchers contributing to this research are from the A*STAR Institute of Molecular and Cell Biology (A*STAR IMCB).
