Designing and validating antisense oligonucleotide therapy for a splicing error mutation of tuberous sclerosis complex 2 gene
Abstract number :
3.2
Submission category :
2. Translational Research / 2E. Other
Year :
2025
Submission ID :
156
Source :
www.aesnet.org
Presentation date :
12/8/2025 12:00:00 AM
Published date :
Authors :
Presenting Author: Seok-Jin Lee, M.D, PhD – Severance Hospital
Ji-Hye Choi, MSc – Yonsei College of Medicine
Ara Ko, M.D, Ph.D. – Severance Hospital
Hoon-Chul Kang, M.D, Ph.D. – Severance Hospital
Chul-Hoon Kim, M.D, Ph.D. – Yonsei College of Medicine
Rationale: Recently, successful therapeutic applications of antisense oligonucleotide (ASO) therapy on various rare diseases have been reported. ASO can be applied to block splicing error mutations. At our tertiary referral center, we have identified a splicing error mutation of tuberous sclerosis complex 2 gene in one of our TSC patients, which according to our in-silico analysis, was considered as an amenable target of splicing modifying antisense oligonucleotide therapy. Tuberous sclerosis can cause multi-systemic manifestations throughout one’s lifetime ranging from skin manifestations, cortical tubers, renal angiomyolipoma, lymphangioleiomyomatosis to name a few. It will be thus important to validate the therapeutic potential of ASO using an appropriate platform ex vivo before applying directly to the patient. Hence, we have decided to embark upon applying antisense oligonucleotide therapy for this mutation with consent from both the patient and the patient’s guardian.
Methods: As one of the tertiary referral hospitals with several hundreds of TSC patients in Korea, we have initially looked for a mutation that could be a possible target of splicing modifying ASO therapy. We have found one female patient with a splicing error mutation. Our in-silico analysis using various tools have indicated that ASO therapy could be feasible in this particular mutation. We have designed more than twenty ASOs to target the splicing error mutation in our patient. These ASOs were tested in the patient-derived iPSCs to see if splicing mutation is corrected by ASO and thus the expression of correct TSC2 mRNA can be restored.
Results: We have identified a splicing error mutation in one of our TSC patients, which causes a cryptic splicing acceptor site thus resulting in the introduction of a pseudoexon with a premature stop codon. Out of more than twenty ASOs we designed, several ASOs have shown promising results to block the cryptic splicing acceptor site thus blocking the transcription of pseudoexon and restoring the normal mRNA expression of TSC2 in our patient-derived iPSC line. Reverse transcriptase polymerase chain reaction analysis has validated the restored mRNA expression of wild-type TSC2 which was further confirmed by Sanger sequencing the mRNA product. Detailed analysis will be presented during a poster session. We are now designing further experiments to validate the effect of ASO therapy using patient-derived organoids.
Conclusions: Our results so far demonstrate that ASO therapy could be applied to a splicing mutation in a TSC patient to restore the correct transcription of TSC2.
Funding: This work has been supported by Korea Health Industry Development Institute
Translational Research