Alessandro Marrero-Gagliardi
TDP-43 is predominantly nuclear RNA binding protein, central in ALS/TDF pathogenesis, with essential functions in RNA metabolism (C. Ling et al., 2013). In pathology TDP-43 mislocalizes and aggregates in the cytoplasm (Ederle & Dormann, 2017). Nuclear loss of TDP-43 induces a multitude of splicing changes, including the de-repression of intronic sequences that are aberrantly incorporated in mature RNA – named cryptic exons (CE) (J. P. Ling et al., 2015). CEs play a key role in ALS pathogenesis and progression, promoting the degradation of genes involved in synaptic vesicle release (Ma, X. et al., 2022; Brown, A. L. et al., 2022) or axonal growth (Baughn, M. W. et al., 2023) among others. Numerous groups develop antisense oligonucleotides (ASOs) targeting individual splicing events as a therapeutic strategy, requiring repeated dosing throughout the patient’s life. Our approach instead uses modified therapeutic U7 (tU7) snRNA genes packaged into a viral vector to enable long-term expression of therapeutic antisense sequences from a single dose. tU7s form part of a stable small nuclear ribonucleoprotein (snRNP) complexes, wich have compact size and accumulate in the nucleus without causing toxic effects. Here, we are able to correct cryptic splicing in transcripts of genes with fundamental roles in neurodegeneration such as: synaptic vesicles release (UNC13A), axonal growth (STMN2), stress granule assembly (G3BP1), autophagy (ATG4B), protein synthesis (AARS1), and to reduce production of a cryptic peptide expressed in CSF of ALS/FTLD-TDP patients (HDGFL2). Importantly, placing special emphasis on the design of a combined tU7 with which we could rescue several CEs with a single therapeutic dose. Thus, we demonstrate a stable and efficient approach to rescue cryptic splicing events caused by loss of TDP-43 in multiple target genes by designing and lentivirally delivering tU7 snRNAs into iPSC-derived lower motor neurons (i3LMNs) (Fernandopulle M. et al., 2018).
