Dual-targeting snRNA gene therapy rescues STMN2 and UNC13A splicing in TDP-43 proteinopathies

Gomberg, T.; Elmsaouri, S.; Kopalle, H. M.; Baughn, M. W.; Beccari, M. S.; McAlonis-Downes, M.; Artates, J. W.; Pant, D.; Mak, H.; Smargon, A. A.; Sander, T. C.; Garcia, E.; Lee, D. P.; Cleveland, D. W.; Yeo, G. W.

doi:10.64898/2025.12.01.691001

bioRxiv · 10.64898/2025.12.01.691001

Dual-targeting snRNA gene therapy rescues STMN2 and UNC13A splicing in TDP-43 proteinopathies

Gomberg, T.·Elmsaouri, S.·Kopalle, H. M.·Baughn, M. W.·Beccari, M. S.·McAlonis-Downes, M.·Artates, J. W.·Pant, D.·Mak, H.·Smargon, A. A.·Sander, T. C.·Garcia, E.·Lee, D. P.·Cleveland, D. W.·Yeo, G. W.

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Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder caused by the selective deterioration of motor neurons in the central nervous system (CNS). A key driver of this pathogenesis is nuclear loss of ALS-associated protein TDP-43, leading to mis-splicing of TDP-43 targets including important neuronal genes STMN2 and UNC13A. Here, we have developed a gene therapy strategy for ALS and related TDP-43 proteinopathies, to correct mis-splicing of both STMN2 and UNC13A cryptic exons using small nuclear RNAs (snRNAs) encoded from a single vector. We identified promoter sequence elements to increase therapeutic snRNA expression by 10-fold, then further optimized the expression cassette with combinatorial snRNA targeting to rescue multiple cryptic splicing targets. The engineered snRNAs restored normal pre-mRNA processing of both STMN2 and UNC13A transcripts despite TDP-43 loss of function, rescuing stathmin-2 protein levels in iPSC derived motor neurons, restoring their axonal regeneration capacity to wild-type levels. In addition, adeno-associated virus (AAV) delivery of the snRNAs to the murine central nervous system in the constitutive cryptic splicing model Stmn2Hum{Delta}GU fully restored cortical Stmn2 pre-mRNA processing, highlighting the utility of snRNAs as a therapeutic modality in vivo. Together, this study demonstrates that snRNAs are a promising and versatile therapeutic strategy for the simultaneous correction of multiple aberrant transcripts affected by cryptic splicing in TDP-43 proteinopathies.

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Gomberg, T., Elmsaouri, S., Kopalle, H. M., Baughn, M. W., Beccari, M. S., McAlonis-Downes, M., Artates, J. W., Pant, D., Mak, H., Smargon, A. A., Sander, T. C., Garcia, E., Lee, D. P., Cleveland, D. W., Yeo, G. W.. 2025-12-03. Dual-targeting snRNA gene therapy rescues STMN2 and UNC13A splicing in TDP-43 proteinopathies. https://doi.org/10.64898/2025.12.01.691001

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