Project summary/abstract (30 lines) Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is a rare genetic metabolic disorder caused by ALDH5A1 mutations. ALDH5A1 encodes SSADH essential for the catabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). In SSADHD, pathologic accumulation of GABA and metabolite γ-hydroxybutyrate (GHB) leads to broad spectrum encephalopathy. Paradoxically, despite heightened ambient GABA, patients with SSADHD are susceptible to seizures and sudden unexpected death in epilepsy (SUDEP), highlighting the significance of compensatory down-regulation of GABA receptors over pathologic GABA build-up. A major unmet medical need for SSADHD is treatment directly addressing the underlying enzyme deficiency such as gene restoration therapy. Proof-of-concept aldh5a1 restoration via adeno-associated virus (AAV) increased survival of SSADH-deficient aldh5a1lox-STOP mice and reversed SSADHD-relevant phenotypes. Furthermore, restoration of ~15% SSADH in relevant cell types is sufficient for enhanced survival. An AAV encompassing a human ALDH5A1 full-length native promoter (FLnP) driving a functional recombinant ALDH5A1 gene is a potential cure for patients with SSADHD. However, it is unclear whether AAV-FLnP-ALDH5A1 drives sufficient functional SSADH expressions in patient cells, and whether systemic AAV delivery (requiring high AAV dose) might lead to therapeutically meaningful brain target engagement. We propose to use patient induced pluripotent stem cells (iPSC)-derived neurons and the SSADH-deficient aldh5a1lox-STOP mouse model to study efficacy and safety of this novel AAV-FLnP-ALDH5A1 construct. R61 Aim 1: Measure SSADH restoration, cultured medium GHB content, and functional properties of patient iPSC-derived neurons. Go-no-go: At least 50% SSADH restored, functional phenotype returned to control. R61 Aim 2: Measure SSADH restoration in brain tissues, GABA receptors, GHB content in blood, electrographic seizures, and survival in SSADH-deficient aldh5a1lox-STOP mice. Go-no-go: GHB reduced to <15µM, reduced seizures and two-fold lifespan extension. R33 Aim 3: Utilize dose de-escalation to determine minimal AAV-FLnP-ALDH5A1 (in AAV9 serotype) threshold for therapeutic effects, and long-term toxicity in SSADH-deficient mice. The project goal is two-fold: 1) Provision of molecular insights into whether AAV-FLnP-ALDH5A1 is a viable clinical candidate for SSADH gene therapy. The proposed study provides necessary insights into whether this novel AAV construct is sufficient for phenotypic reversal in patient-derived iPSC, and whether this gene expression cassette FLnP-ALDH5A1 is effective in various cell types including excitatory and inhibitory neurons. 2) Establishment of practical AAV dosage for therapeutic SSADH gene restoration therapy. The proposed study provides necessary insights into minimal threshold for systemic delivery of translatable AAV serotype leading to brain-wide coverage and associated phen...