Repair of Mutated NF1 mRNA with Trans-Splicing Group I Intron Ribozymes

Therapeutic strategies for Neurofibromatosis Type I (NF1) that correct the underlying pathogenic NF1 variant hold promise for restoring neurofibromin function, reducing tumor burden, and improving patient outcomes by addressing the root cause of the disease rather than its symptoms. Beyond gene editing, transcript reprogramming via RNA trans-splicing has gained attention, particularly with the recent FDA approval of two trans-splicing-based drugs for IND phase 1/2a trials. This study tests whether trans-splicing group I intron ribozymes from Tetrahymena thermophila can be used to repair pathogenic variants of NF1 (pre-)mRNA by 3'-tail replacement.

Splice sites on the NF1 mRNA were identified computationally and validated biochemically, and an efficiency-enhancing Extended Guide Sequence (EGS) of the corresponding ribozyme was identified in a combinatorial experiment.

The correct trans-splicing product of this ribozyme was validated in HEK293 NF1-/- cells expressing mNf1.

This study established a splice site and activity-enhancing extended guide sequences for the repair of NF1 mRNA. Further optimization of the ribozyme, as well as improved delivery methods, may establish ribozyme-based RNA repair as a viable strategy for NF1 treatment.