Abstract
Adeno-associated virus (AAV) vectors have been successfully used to deliver genes for treating rare diseases. However, the systemic administration of high AAV vector doses triggers several adverse effects, including immune response, the asymptomatic elevation of liver transaminase levels, and complement activation. Thus, improving AAV transduction and reducing AAV dosage for treatment is necessary. Recently, we found that a phosphodiesterase-5 inhibitor significantly promoted AAV9 transduction in vitro by regulating the caveolae and macropinocytosis pathways. When AAV9-Gaussian luciferase (AAV9-Gluc) and AAV9-green fluorescent protein (AAV9-GFP) were injected intravenously into mice pre-treated with sildenafil, the expressions of Gluc in the plasma and GFP in muscle tissues significantly increased (P < 0.05). Sildenafil also improved Evans blue permeation in tissues. Additionally, we found that sildenafil promoted Treg proliferation, inhibited B-cell activation, and decreased anti-AAV9 IgG levels (P < 0.05). Furthermore, sildenafil significantly promoted Duchenne muscular dystrophy gene therapy efficacy using AAV9 in mdx mice; it increased micro-dystrophin gene expression, forelimb grip strength, and time spent on the rotarod test, decreased serum creatine kinase levels, and ameliorated histopathology by improving muscle cell morphology and reducing fibrosis (P < 0.05). These results show that sildenafil significantly improved AAV transduction, suppressed the levels of anti-AAV9 IgG, and enhanced the efficacy of gene therapy.
Similar content being viewed by others
Data availability
The data that support the findings of this study are available from the corresponding authors, XW or XX, upon reasonable request.
References
Mingozzi F, High KA. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet. 2011;12:341–55.
Vandendriessche T, Thorrez L, Acosta-Sanchez A, Petrus I, Wang L, Ma L, et al. Efficacy and safety of adeno-associated viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia B gene therapy. J Thromb Haemost. 2007;5:16–24.
Nathwani AC. Gene therapy for hemophilia. Hematol Am Soc Hematol Educ Program. 2019;2019:1–8.
Nagree MS, Scalia S, McKillop WM, Medin JA. An update on gene therapy for lysosomal storage disorders. Expert Opin Biol Ther. 2019;19:655–70.
Duan D. Systemic AAV micro-dystrophin gene therapy for Duchenne muscular dystrophy. Mol Ther. 2018;26:2337–56.
Yiu EM, Kornberg AJ. Duchenne muscular dystrophy. J Paediatr Child Health. 2015;51:759–64.
Kodippili K, Hakim CH, Pan X, Yang HT, Yue Y, Zhang Y, et al. Dual AAV gene therapy for duchenne muscular dystrophy with a 7-kb mini-dystrophin gene in the canine model. Hum Gene Ther. 2018;29:299–311.
Manno CS, Chew AJ, Hutchison S, Larson PJ, Herzog RW, Arruda VR, et al. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood. 2003;101:2963–72.
McIntosh J, Lenting PJ, Rosales C, Lee D, Rabbanian S, Raj D, et al. Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant. Blood. 2013;121:3335–44.
Khabou H, Cordeau C, Pacot L, Fisson S, Dalkara D. Dosage thresholds and influence of transgene cassette in adeno-associated virus-related toxicity. Hum Gene Ther. 2018;29:1235–2341.
Chandler LC, Barnard AR, Caddy SL, Patrício MI, McClements ME, Fu H, et al. Enhancement of adeno-associated virus-mediated gene therapy using hydroxychloroquine in murine and human tissues. Mol Ther Methods Clin Dev. 2019;14:77–89.
Cui S, Ganjawala TH, Abrams GW, Pan ZH. Effect of proteasome inhibitors on the AAV-mediated transduction efficiency in retinal bipolar cells. Curr Gene Ther. 2020;19:404–12.
Pérez-Iturralde A, Carte B, Aldabe R. Consequences of mammalian target of rapamycin inhibition on adeno-associated virus hepatic transduction efficacy. Hum Gene Ther. 2021;32:1242–50.
Chai Z, Zhang X, Rigsbee KM, Wang M, Samulski RJ, Li C. Cryoprecipitate augments the global transduction of the adeno-associated virus serotype 9 after a systemic administration. J Control Release. 2018;286:415–24.
Boolell M, Allen MJ, Ballard SA, Gepi-Attee S, Muirhead GJ, Naylor AM, et al. Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impot Res. 1996;8:47–52.
Michel CC. Capillaries, caveolae, calcium and cyclic nucleotides: a new look at microvascular permeability. J Mol Cell Cardiol. 1998;30:2541–6.
Nagata K, Marbán E, Lawrence JH, Donahue JK. Phosphodiesterase inhibitor-mediated potentiation of adenovirus delivery to myocardium. J Mol Cell Cardiol. 2001;33:575–80.
Nelson MD, Rader F, Tang X, Tavyev J, Nelson SF, Miceli MC, et al. PDE5 inhibition alleviates functional muscle ischemia in boys with Duchenne muscular dystrophy. Neurology. 2014;82:2085–91.
Kniotek M, Boguska A. Sildenafil can affect innate and adaptive immune system in both experimental animals and patients. J Immunol Res. 2017;2017:4541958.
Tannous BA. Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat Protoc. 2009;4:582–91.
Putten van M, Putker K, Overzier M, Adamzek WA, Pasteuning-Vuhman S, Plomp JJ, et al. Natural disease history of the D2-mdx mouse model for Duchenne muscular dystrophy. FASEB J. 2019;33:8110–24.
Murrey DA, Naughton BJ, Duncan FJ, Meadows AS, Ware TA, Campbell KJ, et al. Feasibility and safety of systemic rAAV9-hNAGLU delivery for treating mucopolysaccharidosis IIIB: toxicology, biodistribution, and immunological assessments in primates. Hum Gene Ther Clin Dev. 2014;25:72–84.
Zhong C, Jiang W, Wang Y, Sun J, Wu X, Zhuang Y, et al. Repeated systemic dosing of adeno-associated virus vectors in immunocompetent mice after blockade of T cell costimulatory pathways. Hum Gene Ther. 2022;33:290–300.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402–8.
Zhang C, Liu Q, Dong F, Li L, Du J, Xie Q, et al. Catalpol downregulates vascular endothelial-cadherin expression and induces vascular hyperpermeability. Mol Med Rep. 2016;13:373–8.
Zych M, Roszczyk A, Kniotek M, Kaleta B, Zagozdzon R. Sildenafil citrate influences production of TNF-α in healthy men lymphocytes. J Immunol Res. 2019;2019:8478750.
Hasegawa S, Ito M, Fukami M, Hashimoto M, Hirayama M, Ohno K. Molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice. Redox Rep. 2017;22:26–34.
Masat E, Pavani G, Mingozzi F. Humoral immunity to AAV vectors in gene therapy: challenges and potential solutions. Discov Med. 2013;15:379–89.
Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet. 2020;21:255–72.
Zhu H, Wang T, John Lye R, French BA, Annex BH. Neuraminidase-mediated desialylation augments AAV9-mediated gene expression in skeletal muscle. J Gene Med. 2018;20:e3049.
Chai Z, Zhang X, Dobbins AL, Rigsbee KM, Wang B, Samulski RJ, et al. Optimization of dexamethasone administration for maintaining global transduction efficacy of adeno-associated virus serotype 9. Hum Gene Ther. 2019;30:829–40.
Greish K, Fateel M, Abdelghany S, Rachel N, Alimoradi H, Bakhiet M, et al. Sildenafil citrate improves the delivery and anticancer activity of doxorubicin formulations in a mouse model of breast cancer. J Drug Target. 2018;26:610–5.
Hu J, Ljubimova JY, Inoue S, Konda B, Patil R, Ding H, et al. Phosphodiesterase type 5 inhibitors increase Herceptin transport and treatment efficacy in mouse metastatic brain tumor models. PLoS ONE. 2010;5:e10108.
Bartlett JS, Wilcher R, Samulski RJ. Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors. J Virol. 2000;74:2777–85.
Sanlioglu S, Benson PK, Yang J, Atkinson EM, Reynolds T, Engelhardt JF. Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by rac1 and phosphatidylinositol-3 kinase activation. J Virol. 2000;74:9184–96.
Nonnenmacher M, Weber T. Adeno-associated virus 2 infection requires endocytosis through the CLIC/GEEC pathway. Cell Host Microbe. 2011;10:563–76.
Saghizadeh M, Kramerov AA, Yaghoobzadeh Y, Hu J, Ljubimova JY, Black KL, et al. Adenovirus-driven overexpression of proteinases in organ-cultured normal human corneas leads to diabetic-like changes. Brain Res Bull. 2010;81:262–72.
Hester J, Schiopu A, Nadig SN, Wood KJ. Low-dose rapamycin treatment increases the ability of human regulatory T cells to inhibit transplant arteriosclerosis in vivo. Am J Transplant. 2012;12:2008–16.
Josefowicz SZ, Lu LF, Rudensky AY. Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol. 2012;30:531–64.
Hunter CE, Hosfield BD, Mesfin FM, Pecoraro AR, Liu JY, Shelley WC, et al. Sildenafil attenuates intestinal injury in necrotizing enterocolitis independently of endothelial nitric oxide synthase. J Pediatr Surg. 2022;57:967–73.
Katwal AB, Konkalmatt PR, Piras BA, Hazarika S, Li SS, John Lye R, et al. Adeno-associated virus serotype 9 efficiently targets ischemic skeletal muscle following systemic delivery. Gene Ther. 2013;20:930–8.
Islam BN, Sharman SK, Hou YL, Bridges AE, Singh N, Kim S, et al. Sildenafil suppresses inflammation-driven colorectal cancer in mice. Cancer Prev Res. 2017;10:377–88.
Yiu EM, Kornberg AJ. Duchenne muscular dystrophy. Neurol India. 2008;56:236–47.
Koeks Z, Bladen CL, Salgado D, Zwet van E, Pogoryelova O, McMacken G, et al. Clinical outcomes in Duchenne muscular dystrophy: a study of 5345 patients from the TREAT-NMD DMD global database. J Neuromuscul Dis. 2017;4:293–306.
Ronzitti G, Gross DA, Mingozzi F. Human immune responses to adeno-associated virus (AAV) vectors. Front Immunol. 2020;11:670.
Potter RA, Griffin DA, Heller KN, Peterson EL, Clark EK, Mendell JR, et al. Dose-escalation study of systemically delivered rAAVrh74.MHCK7.micro-dystrophin in the mdx Mouse Model of Duchenne Muscular Dystrophy. Hum Gene Ther. 2021;32:375–89.
Acknowledgements
We would like to thank Editage (www.editage.cn) for English language editing.
Funding
This work was supported by the Project of Science and Technology Department of Sichuan Province, China (grant number: 18SYXHZ0024), the National Natural Science Foundation of China (grant number: 31901052, 81970171, 82070139), and the Shanghai Committee of Science and Technology, China (grant number: 20ZR1415000).
Author information
Authors and Affiliations
Contributions
Conceptualization, XW and XX, Methodology, KZ, XW and XX; Investigation, KZ, MY, JS; Writing-Original Draft, KZY; Writing-review & editing, KZ, MY and XW; Funding acquisition, XW, JZ, and LZ.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Ethical approval
All experimental procedures involving animals were approved by the Ethical Committee of the Shanghai Model Organisms Center, Inc. (IACUC:2022-0001-06).
Consent for publication
All the listed authors have participated in the study, and have seen and approved the submitted manuscript.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Zhou, K., Yuan, M., Sun, J. et al. Sildenafil increases AAV9 transduction after a systemic administration and enhances AAV9-dystrophin therapeutic effect in mdx mice. Gene Ther 31, 19–30 (2024). https://doi.org/10.1038/s41434-023-00411-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41434-023-00411-3
- Springer Nature Limited