Lung-tropic dual AAV-SP-C and microRNA gene therapy attenuates lung injury in mutant Sftpc mice

Mutations in the surfactant protein C (SP-C) gene (SFTPC) impair surfactant homeostasis, leading to respiratory distress in newborns or progressive interstitial lung disease. The most frequent mutation, I73T, results in the expression of a toxic dominant-negative form of SP-C, insinuating that for gene replacement therapy to be successful, suppression of the toxic form of the SP-C protein may also be necessary. Here, we capitalized on our rationally designed lung-tropic adeno-associated virus (AAV)6.2FF vector to develop a combinatorial gene therapy approach for treating SP-C disorders. In I73T-knockin mice exhibiting decreased Sftpc expression and toxic prosurfactant protein C (proSP-C) accumulation resulting in focal airspace enlargement, gene replacement therapy via airway delivery of AAV6.2FF expressing SP-C restored wild-type (WT) Sftpc and mature SP-C protein expression while significantly improving lung function and focal airspace enlargement. Next, we developed a dual-function AAV6.2FF to express functional SP-C and suppress the toxic 173T SFTPC gene (AAV-SPC-miR). The dual-function AAV6.2FF-SP-C-miR decreased endogenous Sftpc expression, restored WT Sftpc, re-expressed the mature SP-C protein without significant proSP-C protein accumulation, and attenuated airspace enlargement. These findings suggest that combination gene therapy is feasible and represents a promising tool for treating SP-C deficiencies and SFTPC mutation-linked lung diseases in humans.