1551 - Directly Reprogrammed Patient Neurons as a Cross-Disease Platform for Neuromuscular Pathogenesis and Gene-Therapy Response

To establish a rapid, patient-derived neuronal assay that (i) recapitulates cellular phenotypes across gene-defined neuromuscular disorders and (ii) quantifies in-vitro pharmacodynamic response to genotype-matched gene therapy. Prior work  demonstrated feasibility in two prototypical conditions involving motor neuron pathology.

Design:

Dermal fibroblasts from affected individuals and unaffected donors are directly converted to induced neurons in 7 days using a small-molecule protocol. Neuronal identity is confirmed by TUJ1 and MAP2 immunostaining and by absence of cholinergic markers. Pre-specified quantitative endpoints were neurite length, the fraction of TUJ1-positive neurons with neurites, and neuronal conversion rate (TUJ1+/DAPI). For gene-therapy testing, cultures received genotype-matched AAV9 vectors at a fixed multiplicity of infection; transduction is verified by PCR for vector-derived transcript normalized to a house-keeping gene. Where applicable, disease-protein restoration is evaluated by Western blot. Image analyses are performed on 12 fields per condition across at least three independent differentiations by blinded analysis. Statistics use ANOVA with Dunnett post hoc tests and unpaired t-tests.

Results: Direct conversion produced reproducible neuronal cultures that  displayed mixed inhibitory and glutamatergic identities. Across disorders, most patient-derived neurons show shortened neurites, a reduced fraction of neurite-bearing neurons, and in some lines decreased conversion rate compared with controls. Genotype-matched AAV9 treatment achieves robust transduction and yields dose-responsive improvements across the same endpoints. In selected cases harboring variants of uncertain significance, phenotypic rescue after gene replacement supported pathogenicity assessment.

Conclusions:

: Directly reprogrammed patient neurons constitute a fast, scalable, and disease-agnostic human model; an important avenue for neuromuscular research. The assay captures core cellular pathology, delivers quantifiable pharmacodynamic readouts for vector/dose ranking, and aids variant interpretation, thereby accelerating translation from mechanism to early-phase clinical studies