Nutritional Genomics Uncovers Vitamin B3 Therapy as Curative for NAXD Disease

Vitamins are essential metabolites that must be obtained from external sources. All 13 vitamins were identified in the early 1900s, a rich era of biochemistry. More recently, they are consumed indiscriminately. We introduce a new nutritional genomics framework that begins with a treatment and identifies all potential disease candidates. Using a genome-wide CRISPR screen at varying vitamin B3 levels, we nominated dozens of diseases for B3-based therapy. The top candidate was NAXD, a proofreading enzyme involved in correcting an aberrant form of NADH (NADHX). Mutations in NAXD cause severe neurodevelopmental disease, with no proven therapies. We created a new mouse model of disease, observing NADHX accumulation and NAD+ depletion in affected organs, leading to impaired serine biosynthesis. Low B3 diets worsened the disease, while B3 supplementation was curative, extending lifespan by more than 20-fold. This work introduces a new approach to nutritional genomics and precision vitamin therapies. Overall design: Nuclei were isolated from frozen hemi brains of P5 mice across different genotypes and experimental treatments. All mouse pups were treated with either vehicle or 750 mg/kg Nicotinamide Riboside starting from postnatal day 0 until collection on postnatal day 5. Mice were perfused with PBS, and the left hemi-brains (excluding the olfactory bulb) were collected from both Control and NAXD KO mice (n = 4 per genotype per treatment group). We then employed a single-nucleus RNA sequencing approach on these isolated nuclei using the 10x Genomics GEM-X Flex Kit protocol, where samples first undergo fixation before Gene Expression library preparation. This single-nucleus RNA-seq experiment using the GEM-X Flex protocol is probe-based rather than using 3' capture. We randomized the samples being assigned to each mouse probe barcode and assigned to each GEM reaction.