HMGB1-targeted anti-inflammatory rBox A gene therapy for viral airway infections and systemic inflammatory disease models

High mobility group box 1 (HMGB1) is a host-derived proinflammatory molecule released during cell injury. Elevated extracellular HMGB1 levels have been associated with disease severity. HMGB1 contributes to host influenza responses by stimulating TLR4/MD-2 signaling, amplifying lung inflammation, and tissue damage via cytokine production and leukocyte infiltration. HMGB1 also plays a central role in other inflammatory diseases, including sepsis and liver toxicity, making it both a biomarker of disease severity and a potential therapeutic target. We previously reported development of a non-replicating adenovirus (AdV) vector that carries an inflammation-inducible cassette that remains dormant in the absence of inflammation but produces recombinant HMGB1 Box A, a competitive antagonist of HMGB1, in response to inflammatory stimuli. Therapeutic treatment with AdV.C3-Tat/HIV-Box A mitigated lung and systemic inflammation in response to influenza in mice and cotton rats. Herein, we have optimized vector delivery and expanded its use in multiple models of infectious and non-infectious HMGB1-induced inflammation. IMPORTANCE: Despite promising preclinical outcomes, clinical translation of high mobility group box 1 (HMGB1) or TLR4/MD-2 antagonists for inflammatory diseases has been limited by challenges of drug synthesis, dosing, delivery, and off-target effects. Recombinant Box A (rBox A) is a competitive inhibitor of HMGB1 that binds to TLR4 and disrupts HMGB1-mediated TLR4/MD-2 signaling. rBox A exhibits broad anti-inflammatory activity across multiple diseases (e.g., endotoxemia, sepsis, neuroimmune disorders, ischemia/reperfusion); however, its therapeutic utility is constrained by the need for high, repeated dosing. We have engineered a non-replicating adenoviral (AdV) vector (AdV.C3-Tat/HIV-Box A) that produces recombinant (r)Box A when "sensed" by an inflammation-responsive complement C3 (C3) promoter. Therapeutic administration of AdV.C3-Tat/HIV-Box A resulted in significant protection and reduced inflammation in multiple HMGB1-mediated disease models, advancing a first-in-class, broadly applicable, host-directed gene therapy that selectively disrupts HMGB1-TLR4 signaling and has potential as both a broad-spectrum therapeutic for HMGB1-driven inflammatory diseases and a rapid-response countermeasure against emerging respiratory pathogens.