Abstract
Surfactant protein-B (SP-B) deficiency is a lethal neonatal respiratory disease with few therapeutic options. Gene therapy using adeno-associated viruses (AAVs) to deliver human SFTPB cDNA (AAV-hSPB) can improve survival in a mouse model of SP-B deficiency. However, the effect of this gene therapy wanes. Gene therapy efficacy could be prolonged if AAV vectors could be redosed, but readministering vectors is hindered by an immune response that includes TLR9 (Toll-like receptor 9) recognition of unmethylated CpG DNA motifs in the AAV genome. One strategy to mitigate TLR9 recognition of AAV is to incorporate decoy nucleotide sequences within the AAV genome. In this work we examined if an AAV containing these TLR9 inhibitory oligonucleotide sequences (AAV-hSPB(TLR9i)) could mitigate the immune response sufficiently to redose AAV in the lungs and prolong the survival of SP-B-deficient mice. Indeed, AAV-hSPB(TLR9i) could be redosed multiple times, which significantly improved survival in our mouse model. This was partially a result of long-term increased SFTPB RNA and SP-B protein expression. Conversely, redosing AAV-hSPB resulted in the rapid death of SP-B-deficient mice after the second AAV dose. TLR9 inhibition enabled readministration by avoiding the broad stimulation of genes belonging to multiple pathways in the host immune and inflammatory responses, including components of the IFN pathways. Thus, redosing of AAV vectors in the lungs using TLR9 inhibitory sequences is a promising strategy for prolonging gene therapy efficacy, with a proof of concept for AAV readministration in a clinically relevant mouse model of SP-B deficiency.