David Thompson

Amyotrophic lateral sclerosis (ALS) caused by mutation in superoxide dismutase 1 ( SOD1 ) accounts for 15-30% of familial ALS and is typically autosomal dominant. How single base pair/amino acid changes in this small protein cause neurodegeneration is unknown. In North America, SOD1 ^A4V is the most common familial ALS SOD1 mutation and results in an aggressive form of ALS. Here, we present a novel genomically humanised mouse model of SOD1 ^A4V , in which the mouse Sod1 locus has been replaced by the human SOD1 gene, with intact genomic architecture of exons and introns, but bearing an A4V mutation. In agreement with previously reported human genomic knock-in mice, the phenotype is mild; however, transcriptomic and metabolomic profiling reveal significant dysregulation of glycolysis, the tricarboxylic acid (TCA) cycle, and lipid metabolism. These changes suggest an early bioenergetic imbalance that precedes neuromuscular impairment. Our findings support metabolic dysfunction as an early event in ALS pathogenesis. This freely available SOD1 ^A4V model provides a valuable tool for studying ALS progression and identifying therapeutic targets for pre-symptomatic treatment. SUMMARY STATEMENT: This study describes the generation and analysis of novel genomically humanised SOD1 ^A4V mice, revealing metabolic dysfunction through integrated multi-omic analyses, characterising a freely available potential pre-symptomatic ALS model for future research.

Loss of function of the RNA-binding protein TDP-43 (TDP-LOF) is a hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Here we describe TDP-REG, which exploits the specificity of cryptic splicing induced by TDP-LOF to drive protein expression when and where the disease process occurs. The SpliceNouveau algorithm combines deep learning with rational design to generate customizable cryptic splicing events within protein-coding sequences. We demonstrate that expression of TDP-REG reporters is tightly coupled to TDP-LOF in vitro and in vivo. TDP-REG enables genomic prime editing to ablate the UNC13A cryptic donor splice site specifically upon TDP-LOF. Finally, we design TDP-REG vectors encoding a TDP-43/Raver1 fusion protein that rescues key pathological cryptic splicing events, paving the way for the development of precision therapies for TDP43-related disorders.