Gene Therapy to Protect the Lung from Oxidant Stress

AbstractThe focus of this proposal is the development of therapy to protect the lung with an extracellular anti oxidant defense to treat chronic obstructive pulmonary diseaseCOPDa chronic disorder in which inhaled oxidants from tobacco smokepollution and generated within the lung by activated inflammatory cells perpetuate injury to the lung epithelium and endotheliumThe LEXEO strategy is to use in vivo gene Otherapy technology to provide a persistent extracellular anti oxidant enzyme shield to the lung that will inactivate superoxideand H Omajor extracellular oxidant stresses to the lungLEXEO will capitalize on an innovative strategy developed by the Crystal laboratory at Weill Cornellin which genes for catalase and superoxide dismutaseSODhave been genetically modified to secrete functional monomeric antioxidant enzymes that can diffuse in the extracellular milieuproviding the lung with an effective extracellular anti oxidant shieldCatalase is a tetrameric intracellular enzyme that is too large to diffuse if designed to be secretedTo use catalase as an effective extracellular anti oxidantthe gene was modified to prevent the wrapping loop domain to mediate tetramer formationWith the addition of a secretory signalthe human catalase monomerhCatWLis secretedcapable of functioning to catalyze extracellular H Oto H OSuperoxide dismutaseSODis secretedbut it is a large tetramerand has a heparin binding domain that attaches it to cell surfacesTo modify SODinto a more effective lung extracellular antioxidanta loop critical for tetramer formation was modified and the heparin binding domain removedhSOD hdresulting in an effective monomer antioxidant enzyme that will not bind to cell surfacesA serotype rhadeno associatedAAVgene transfer vector will be used to genetically modify the liver to express and secrete the modified catalase and or SODmonomersIdentification of the optimal vector for further clinical development in a phasefuture proposal will be carried out instepsin vitroaimand in vivoFour AAVrhcandidates will be evaluatedAAVrhhCatWLexpressing the catalase monomerAAVrhhSOD hdSODmonomerAAVrhhCatWLhSOD hdboth monomersand AAVrhhSODhdhCatWLsame but with SOD hdin thepositionThe deliverable is to identify LEXthe best candidate AAVrhvector that will generate a persistent extracellular antioxidant shield of the lungAimTo compare in vitro the levels of expression of secretedfunctional modified catalase and or SODmediated by AAVrhvectors with the expression cassetteshCatWLhSOD hdhCatWLhSOD hdand hSOD hdhCatWLAimIn an in vivo murine model of COPDquantify the ability of the optimal antioxidant vector from aimto express secretedfunctional modified catalase and SODcapable of protecting lung endothelium and epithelium from chronic exposure to cigarette smoke NarrativeThe focus of this proposal is the development of a gene therapy to protect the lung with an extracellular anti oxidant defense to treat chronic obstructive pulmonary diseaseCOPDa chronic disorder in which inhaled oxidants from tobacco smokepollution and generated within the lung by activated inflammatory cells play a major role in initiating and perpetuating injury to the lung epithelium and endothelium fundamental to pathogenesis of the disease