ABSTRACT This proposal describes a novel concept for acidification of the lysosome and, subsequently, restoration of au- tophagic flux. Autophagy, the intracellular process by which proteins and organelles are degraded, requires an acidic lysosome. Failure to acidify the lysosomal compartment leads to accumulation of autophagosomes, lipids, and associated undigested content. Impaired lysosomal acidification and reduced autophagic flux are central to non-alcoholic fatty liver disease (NAFLD), hereafter referred to by its new nomenclature, metabolic-dysfunction associated steatotic liver disease (MASLD). The risk factors for developing MASLD increase with advancing age and, therefore, not surprisingly, MASLD occurs primarily among the middle-aged and the elderly. Current phar- macological interventions do not target the liver directly to treat MASLD and, instead, affect secondary pathways or mechanisms (e.g., GLP-1 receptor agonists to decrease food intake). Key to our advance is the engineering of a treatment that responds to the pH of a dysfunctional lysosome (pH 5.7 - 6) to release acid and, therein, further lower the pH to a healthy/normal value (pH 4 - 5). This acid-activated therapy, which we call LysoGen (short for “lysosomal regeneration”) is composed of polyester-based co-polymers comprising a potent diacid, tetrafluorosuccinic acid (TFSA). We hypothesize that, upon accumulation in liver lysosomes after intrave- nous administration, LysoGen will release acid, thereby restoring lysosomal acidity and autophagic flux, as well as rescuing functional and histological hallmarks of MASLD, such as improved glucose/insulin tolerance and reduced lipid droplet accumulation in the liver. Furthermore, we hypothesize that the ex- tent of restoration will depend on the LysoGen polymer composition and dosing regimen. Importantly, substantial preliminary data support the proposed studies, well-characterized materials and rigorous experi- mental designs are established, and essential cross-disciplinary collaborations and expertise (polymer chemis- try, biomaterials, cell metabolism, autophagy, and MASLD) are in place to address these hypotheses. LysoGen provides an unprecedented opportunity to treat MASLD with a first-of-its-kind therapy. Herein, we seek to in- crease our understanding of this new technology and its impact on the biology and pathology of the MASLD disease state as well as elucidate and optimize this approach as a prelude to future clinical translation. The specific aims of this five-year proposal include: Aim 1. Synthesize and characterize LysoGen polymers that activate at pH 6 and are targeted to lysosomes; Aim 2. Determine the in vitro bioavailability and resulting bioac- tivity of LysoGen; and, Aim 3. Evaluate the toxicity, biodistribution and efficacy of the optimized LysoGen formu- lation in a high-fat diet (HFD)-induced MASLD mouse model.