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Gene Therapy for Lung Fibrosis with AAV9-SGPL1
Ranjha Khan1, Aritra Bhattacharyya2, Gizachew Tassew1, Babak Oskouian1, Maria L. Allende3, Richard L. Proia3, Joanna Lee1, Xiaoyang Yin2, Mallar Bhattacharya2 and Julie D. Saba1
1Department of Pediatrics, University of California, San Francisco, CA, United States.
2Division of Pulmonary, Critical Care, Allergy, and Sleep, Department of Medicine, UCSF, CA, United States.
3NIDDK, NIH, United States.
Background: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease associated with a survival of 3-5 years. Coronavirus infections including the recent SARS-CoV2 pandemic represent a risk factor for IPF. Two FDA-approved drugs may prolong survival, but only lung transplantation can cure IPF. Thus, new therapeutic strategies are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes pro-inflammatory and pro-fibrotic signaling via TGF-β/SMAD and other pathways. S1P lyase (SPL) catalyzes irreversible S1P degradation and is upregulated in IPF. We hypothesize that adeno-associated virus 9-mediated delivery of the human SPL gene SGPL1 (AAV β/SPL) will raise lung SPL activity, reduce S1P, and prevent IPF progression in the bleomycin mouse model of IPF.
Methods: 8 to 10-week-old C57BL/6 wild-type mice and a new mouse model of SPL insufficiency generated by gene editing called SPLR222Q (Knock-In) mice were administered 3U/kg Bleomycin intratracheally on day 1 to induce pulmonary fibrosis. Some mice from each group received no bleomycin as controls. In some experiments, wild-type mice were treated with 5e11 vg AAV-SPL i.v. on days 12-14. Mice were euthanized on day 21. Epithelial cells were isolated from harvested lungs using a MojoSort Ep-Cam positive selection kit. Immunoblotting and qRT-PCR were performed for target proteins/gene expression analysis. Lung fibrosis was determined by hydroxyproline assay, Sirius Red/Fast green solution, and collagen gene expression. Publicly available scRNA data was analyzed for SGPL1 expression in human and murine lungs. One-way ANOVA or t-test was used for comparison.
Results: Analysis of publicly available scRNAseq data show high expression of SGPL1/Sgpl1 in human and murine lung epithelial cells and upregulation in pulmonary fibrosis. We show Sgpl1 expression is upregulated in lung epithelial cells isolated from bleomycin-injured mice compared to control mice, and both Sgpl1 expression and activity are upregulated in whole lung tissue after bleomycin treatment. SPLR222Q KI mice exhibited higher collagen content, expression of collagen genes, a-SMA, and TGF β signaling than wild-type mice lungs both at baseline and after bleomycin injury. AAV β/SPL increased SPL activity in A549 lung epithelial cells. In vivo, treatment of wild-type mice with AAV-SPL further augmented lung SPL activity and decreased fibrosis and pro-fibrotic signaling compared to saline-treated controls.
Conclusion: Our results provide proof of concept for AAV-SPL as first-in-class single-dose gene therapy with the potential to cure IPF, supplanting partially effective medical treatments and costly lung transplantation.
This work was supported by institutional discretionary funds to MB and the UCSF Catalyst program, NIH S10OD0018070, and the Swim Across America Foundation to JDS.
Speakers
Ranjha Khan