The interactions of a pathogen with the host take place within the confines of infected tissues. However, current technologies do not allow for contextual characterization of infection. We report a unified spatial transcriptomic analysis detecting pathogen- and host-transcripts simultaneously. We showcase our approach using a P. aeruginosa -induced ocular infection, where we observed differential enrichment of host and bacterial transcripts at specific anatomical sites. The ridge regression model, trained using a minimum of 150 host gene features, accurately predicted bacterial burden in tissue regions. The tissue-in-depth gene expression enrichment analysis identified bacterial transcript PA2590, encoding a currently uncharacterized gene as deeply penetrant. Infection experiments with the PA2590 transposon deletion mutant strain caused less disease than the WT strain. Comparative structural analysis identified the PA2590 gene product as an iron-scavenging and cobalamin transporter. Cumulatively, our data highlight coordinated spatial interplay between the host and the pathogen representing an approach to identify novel virulence traits. ### Competing Interest Statement HZ, ON, NC, BJ, EO, CT, GD, HL, OP, and MG are employee of Moderna Inc. and may hold stock/stock options in Moderna, Inc. SA, MM, and EP have no conflicts to declare.

Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade®/modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a.

Propionic acidemia/aciduria (PA) is an ultra-rare, life-threatening, inherited metabolic disorder caused by deficiency of the mitochondrial enzyme, propionyl-CoA carboxylase (PCC) composed of six alpha (PCCA) and six beta (PCCB) subunits. We herein report an enzyme replacement approach to treat PA using a combination of two messenger RNAs (mRNAs) (dual mRNAs) encoding both human PCCA (hPCCA) and PCCB (hPCCB) encapsulated in biodegradable lipid nanoparticles (LNPs) to produce functional PCC enzyme in liver. In patient fibroblasts, dual mRNAs encoded proteins localize in mitochondria and produce higher PCC enzyme activity vs. single (PCCA or PCCB) mRNA alone. In a hypomorphic murine model of PA, dual mRNAs normalize ammonia similarly to carglumic acid, a drug approved in Europe for the treatment of hyperammonemia due to PA. Dual mRNAs additionally restore functional PCC enzyme in liver and thus reduce primary disease-associated toxins in a dose-dependent manner in long-term 3- and 6-month repeat-dose studies in PA mice. Dual mRNAs are well-tolerated in these studies with no adverse findings. These studies demonstrate the potential of mRNA technology to chronically administer multiple mRNAs to produce large complex enzymes, with applicability to other genetic disorders.