Intra-Tracheal Adeno-Associated Virus Mediates Gene Transduction During Static Cold Storage in Rodent Lung Transplantation

Abstract

<h3>Purpose</h3> We tested the efficacy of intra-tracheal Adeno-associated Virus (AAV) to deliver reporter genes during static cold storage (SCS) in rat lung grafts. <h3>Methods</h3> Recombinant AAV subtype-9 vectors encoding firefly luciferase were produced in HEK293 cells (AAV-Luc). Lewis rat lung grafts received either 8e10 viral genomes (VGs) in 100 μL saline ([8e8 VG/μL], N=3), 4e11 VGs in 100 μL saline ([4e9VG/μL], N=3), or 100 μL saline only (N=2) via the trachea during 1 hour of SCS. Syngeneic rats underwent single left lung transplantation. Animals underwent bioluminescence imaging on post-operative weeks 1, 2, 4, and 8, then were sacrificed. Explanted tissues were lysed and incubated with luciferin to quantify reporter activity. Immunohistochemistry was performed using tissue from rats transplanted with lungs treated with 4e11 VGs of AAV encoding Green Fluorescent Protein (GFP) and sacrificed at day 14 (N=2). <h3>Results</h3> Following transplant, in vivo bioluminescence was observed in the left chest of animals that received AAV-Luc (Figure 1A). In vivo signal was similar in both groups (mean radiance 7.6e4 vs 7.1e4 photons per second per square cm per steradian (p/s/cm2/sr) at the terminal time point (p=0.41), and similar to that of control animals (mean 6.1e4 p/s/cm2/sr Figure 1B). In the explanted transplant lung, mean luminescent activity was 9.2e2 relative luminescent units (RLU) per gram tissue in controls, compared to 3.0e4 RLU/g in grafts receiving [8e8 VG/μL] and 2.0e5 RLU/g in grafts receiving [4e9 VG/μL] AAV-Luc (Figure 1C). Immunohistochemistry revealed positive GFP staining primarily in pneumocytes of the AAV-GFP treated lung only (Figure 1D). <h3>Conclusion</h3> Here we demonstrate for the first time successful intra-tracheal AAV-mediated gene transduction during SCS, with subsequent expression in the donor lung for at least 8 weeks without apparent off-target expression. Gene expression appears to be primarily seen in alveolar pneumocytes with this route of administration.