Mini-Catalytically Inactive Cas13X-Derived RNA Base Editing of β-Catenin Attenuates Pulmonary Damage in a Murine Acute Lung Injury Model

Abstract

Acute lung injury (ALI) is characterized by a considerable mortality rate and currently lacks viable therapeutic strategies. Alveolar type II epithelial cells (AT2 cells) play a critical role in lung injury repair, potentially through activation of the Wnt/β-catenin signaling cascade, which may enhance regenerative ability of lung tissue. In this study, we developed a mini-catalytically inactive Cas13X (mini dCas13X)-based adenosine-to-inosine (A-to-I) RNA editing approach, designated as β-catenin T41 editing to treat alveolar type 2 cells (CARTEL), with the objective of alleviating lung damage in ALI. We found that CARTEL proficiently performed base editing on β-catenin, achieving a high A-to-I conversion rate with minimal off-target effects. Moreover, CARTEL significantly inhibited the degradation of β-catenin, amplified Wnt/β-catenin signaling activation and facilitated cellular proliferation. In a murine model of lipopolysaccharide (LPS)-induced ALI, a single adeno-associated virus (AAV)-mediated administration of CARTEL effectively and primarily transduced AT2 cells, resulting in attenuated lung injury, enhanced AT2 cell proliferation, and improved pulmonary function, with no detected long-term risks. Collectively, these findings revealed that CARTEL-mediated RNA editing represents a promising therapeutic strategy to counteract lung injury occurring in diverse settings.

Keywords

β-catenin / acute lung injury / alveolar type II epithelial cells / base editing / CRISPR-Cas13

References

[1] G. Bellani, J. G. Laffey, T. Pham, et al., “Epidemiology, Patterns of Care, and Mortality for Patients With Acute Respiratory Distress Syndrome in Intensive Care Units in 50 Countries,” JAMA 315, no. 8 (2016): 788–800.

[2] M. C. Basil, J. Katzen, A. E. Engler, et al., “The Cellular and Physiological Basis for Lung Repair and Regeneration: Past, Present, and Future,” Cell Stem Cell 26, no. 4 (2020): 482–502.

[3] N. J. Meyer, L. Gattinoni, and C. S. Calfee, “Acute Respiratory Distress Syndrome,” Lancet 398, no. 10300 (2021): 622–637.

[4] C. E. Barkauskas, M. J. Cronce, C. R. Rackley, et al., “Type 2 Alveolar Cells Are Stem Cells in Adult Lung,” Journal of Clinical Investigation 123, no. 7 (2013): 3025–3036.

[5] W. J. Zacharias, D. B. Frank, J. A. Zepp, et al., “Regeneration of the Lung Alveolus by an Evolutionarily Conserved Epithelial Progenitor,” Nature 555, no. 7695 (2018): 251–255.

[6] R. L. Zemans, N. Briones, M. Campbell, et al., “Neutrophil Transmigration Triggers Repair of the Lung Epithelium via Beta-Catenin Signaling,” PNAS 108, no. 38 (2011): 15990–15995.

[7] A. N. Nabhan, D. G. Brownfield, P. B. Harbury, M. A. Krasnow, and T. J. Desai, “Single-Cell Wnt Signaling Niches Maintain Stemness of Alveolar Type 2 Cells,” Science 359, no. 6380 (2018): 1118–1123.

[8] O. O. Abudayyeh, J. S. Gootenberg, B. Franklin, et al., “A Cytosine Deaminase for Programmable Single-Base RNA Editing,” Science 365, no. 6451 (2019): 382–386.

[9] O. O. Abudayyeh, J. S. Gootenberg, S. Konermann, et al., “C2c2 is a Single-Component Programmable RNA-Guided RNA-Targeting CRISPR Effector,” Science 353, no. 6299 (2016): aaf5573.

[10] C. Xu, Y. Zhou, Q. Xiao, et al., “Programmable RNA Editing With Compact CRISPR-Cas13 Systems From Uncultivated Microbes,” Nature Methods 18, no. 5 (2021): 499–506.

[11] B. L. Ellis, M. L. Hirsch, J. C. Barker, and J. P. Connelly, “Steininger RJ 3rd, Porteus MH. A Survey of Ex Vivo/in Vitro Transduction Efficiency of Mammalian Primary Cells and Cell Lines With Nine Natural Adeno-associated Virus (AAV1-9) and One Engineered Adeno-Associated Virus Serotype,” Virology Journal 10 (2013): 74.

[12] M. P. Seiler, A. D. Miller, J. Zabner, and C. L. Halbert, “Adeno-Associated Virus Types 5 and 6 Use Distinct Receptors for Cell Entry,” Human Gene Therapy 17, no. 1 (2006): 10–19.

[13] M. P. Limberis, L. H. Vandenberghe, L. Zhang, R. J. Pickles, and J. M. Wilson, “Transduction Efficiencies of Novel AAV Vectors in Mouse Airway Epithelium in Vivo and Human Ciliated Airway Epithelium in Vitro,” Molecular Therapy 17, no. 2 (2009): 294–301.

[14] M. H. Kang, L. P. van Lieshout, L. Xu, et al., “A Lung Tropic AAV Vector Improves Survival in a Mouse Model of Surfactant B Deficiency,” Nature Communications 11, no. 1 (2020): 3929.

[15] Q. Xiao, Z. Xu, Y. Xue, et al., “Rescue of Autosomal Dominant Hearing Loss by in Vivo Delivery of Mini dCas13X-Derived RNA Base Editor,” Science Translational Medicine 14, no. 654 (2022): eabn0449.

[16] H. Clevers, K. M. Loh, and R. Nusse, “Stem Cell Signaling. An Integral Program for Tissue Renewal and Regeneration: Wnt Signaling and Stem Cell Control,” Science 346, no. 6205 (2014): 1248012.

[17] A. N. Nabhan, J. D. Webster, J. J. Adams, et al., “Targeted Alveolar Regeneration With Frizzled-Specific Agonists,” Cell 186, no. 14 (2023): 2995–3012.e15.

[18] H. Chen, C. Lu, S. J. Lee, and Y. Li, “Protocol to Generate and Characterize Potent and Selective WNT Mimetic Molecules,” STAR Protocol 1, no. 1 (2020): 100043.

[19] M. Kahn, “Can We Safely Target the WNT Pathway,” Nature Reviews Drug Discovery 13, no. 7 (2014): 513–532.

[20] G. Li, M. Jin, Z. Li, et al., “Mini-dCas13X-Mediated RNA Editing Restores Dystrophin Expression in a Humanized Mouse Model of Duchenne Muscular Dystrophy,” Journal of Clinical Investigation 133, no. 3 (2023): e162809.

[21] S. Shen, P. Wang, P. Wu, et al., “CasRx-Based Wnt Activation Promotes Alveolar Regeneration While Ameliorating Pulmonary Fibrosis in a Mouse Model of Lung Injury,” Molecular Therapy: The Journal of the American Society of Gene Therapy 32, no. 11 (2024): 3974–3989.

[22] M. G. Kluesner, D. A. Nedveck, W. S. Lahr, et al., “EditR: A Method to Quantify Base Editing From Sanger Sequencing,” CRISPR Journal 1, no. 3 (2018): 239–250.

[23] K. Clement, H. Rees, M. C. Canver, et al., “CRISPResso2 Provides Accurate and Rapid Genome Editing Sequence Analysis,” Nature Biotechnology 37, no. 3 (2019): 224–226.

[24] K. J. Livak and T. D. Schmittgen, “Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2(-Delta Delta C(T)) Method,” Methods 25, no. 4 (2001): 402–408.

[25] G. Matute-Bello, G. Downey, B. B. Moore, et al., “An Official American Thoracic Society Workshop Report: Features and Measurements of Experimental Acute Lung Injury in Animals,” American Journal of Respiratory Cell and Molecular Biology 44, no. 5 (2011): 725–738.

RIGHTS & PERMISSIONS

2026 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.