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

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 <i>STMN2</i> and <i>UNC13A</i> . Here, we have developed a gene therapy strategy for ALS and related TDP-43 proteinopathies, to correct mis-splicing of both <i>STMN2</i> and <i>UNC13A</i> 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 <i>STMN2</i> and <i>UNC13A</i> 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 <i>Stmn2</i> ^HumΔGU fully restored cortical <i>Stmn2</i> pre-mRNA processing, highlighting the utility of snRNAs as a therapeutic modality <i>in vivo</i> . 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.