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

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 Stmn2HumΔGU 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. Competing Interest Statement G.W.Y. is a cofounder, member of the Board of Directors, Scientific Advisory Board member, equity holder, and paid consultant for Eclipse BioInnovations. G.W.Y.'s interests have been reviewed and approved by the University of California San Diego, in accordance with its conflict-of-interest policies. All other authors declare no competing interests.