Efficient AAV8 delivery to the liver via isolated hepatic perfusion and analysis of hepatic lobule transduction patterns

Abstract

Background and aims: Adeno-associated virus (AAV) is becoming an attractive vector due to its low toxicity and minimal immunogenicity. However, liver-targeted AAV gene therapy still faces challenges, such as low delivery efficiency and safety risks associated with high vector doses. Isolated hepatic perfusion (IHP) has been explored as a localized drug delivery method, yet its full potential in gene therapy remains under investigation. Here, we investigated the efficiency of AAV8 delivery via the IHP route and its preference for hepatic transduction.

Methods: The IHP route was established through surgery in rats and cynomolgus monkey, and the AAV8-dTomato solution was injected into the entire liver through the inflow tract and maintained for 10 min. One week later, liver tissues were obtained, and the dTomato fluorescence expression area fraction and intensity were analyzed.

Results: AAV8-dTomato delivery via the IHP resulted in over 60% dTomato-positive areas in rat liver and showed higher efficiency than the portal vein (PV) and inferior vena cava (IVC) routes at equivalent doses. In rats, AAV8-dTomato expression was primarily periportal across IHP, PV, and IVC routes, while in cynomolgus monkey, IHP delivery showed a pericentral pattern.

Conclusions: In this study, we found that IHP is an effective strategy for AAV8 delivery. In addition, the distribution characteristics of AAV8, when delivered in cynomolgus monkey via IHP, provide candidate vector delivery schemes for gene therapy for different types of genetic liver diseases.

Keywords

Gene therapy / Adeno-associated virus (AAV) / Isolated hepatic perfusion (IHP) / Liver zonation / Genetic liver disease

Authors’ contributions

Chao Wang: Writing e original draft, Project administration, Methodology, Data curation. Mingming Fan: Writing e original draft, Project administration. Jianrong Liu: Methodology, Investi-gation. Xiaolu Guo: Data curation. Zhipeng Tan: Visualization, Formal analysis. Xueying Huang: Visualization, Formal analysis. Zhuoran Li: Visualization, Formal analysis. Xiaomei Liu: Visuali-zation. Ye Zhang: Visualization. Jianqi Feng: Investigation. Rui Fang: Investigation. Li Wang: Writing e review & editing, Project administration, Conceptualization, Supervision, Funding acquisi-tion. Qiong Ke: Writing e review & editing, Conceptualization, Supervision, Funding acquisition.

Data availability statement

The datasets used and analyzed during the current study are available from the corresponding authors upon reasonable request.

Declaration of competing interest

The authors declare that there is no conflicts of interest.

Acknowledgements

This work was founded by the National Key Research and Devel-opment Program of China (No. 2022YFA1104100, 2018YFA0801404, 2017YFA0104304, and 2024YFA1107200), The National Natural Sci-ence Foundation of China (No. 82171617 and 82471689), Guangdong Special Support Program (No. 2019BT02Y276), Sanming Project of Medicine in Shenzhen Nanshan (No. SZSM202103012), and Shenzhen Nanshan District Science and Technology Program (No. NS2022023, NS2023012, NSZD2024048, and NSZD2024051).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.livres.2025.04.006.

References

[1] Steffin DHM, Hsieh EM, Rouce RH. Gene therapy: current applications and future possibilities. Adv Pediatr. 2019;66:37e54. https://doi.org/10.1016/j.yapd.2019.04.001.

[2] Naso MF, Tomkowicz B, Perry WL, Strohl WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs. 2017;31:317e334. https://doi.org/10.1007/s40259-017-0234-5.

[3] Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019;18:358e378. https://doi.org/10.1038/s41573-019-0012-9.

[4] Li N, Gou S, Wang J, et al. CRISPR/Cas9-mediated gene correction in newborn rabbits with hereditary tyrosinemia type I. Mol Ther. 2021;29:1001e1015. https://doi.org/10.1016/j.ymthe.2020.11.023.

[5] Villiger L, Grisch-Chan HM, Lindsay H, et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat Med. 2018; 24: 1519e1525.https://doi.org/10.1038/s41591-018-0209-1.

[6] Ryu SM, Koo T, Kim K, et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat Biotechnol. 2018; 36: 536e539. https://doi.org/10.1038/nbt.4148.

[7] Meyer NL, Chapman MS. Adeno-associated virus (AAV) cell entry: structural insights. Trends Microbiol. 2022;30:432e451. https://doi.org/10.1016/j.tim.2021.09.005.

[8] Rodríguez-Márquez E, Meumann N, Büning H. Adeno-associated virus (AAV) capsid engineering in liver-directed gene therapy. Expert Opin Biol Ther. 2021;21:749e766. https://doi.org/10.1080/14712598.2021.1865303.

[9] Balestra D, Ferrarese M, Lombardi S, et al. An exon-specific small nuclear U1 RNA (ExSpeU1) improves hepatic OTC expression in a splicing-defective spf/ash mouse model of ornithine transcarbamylase deficiency. Int J Mol Sci. 2020; 21: 8735. https://doi.org/10.3390/ijms21228735.

[10] Tan R, Li J, Liu L, et al. CSAD ameliorates lipid accumulation in high-fat diet-fed mice. Int J Mol Sci. 2022; 23:15931. https://doi.org/10.3390/ijms232415931.

[11] Hinderer C, Katz N, Buza EL, et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Hum Gene Ther. 2018;29:285e298. https://doi.org/10.1089/hum.2018.015.

[12] Nguyen GN, Everett JK, Kafle S, et al. A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells. Nat Biotechnol. 2021;39:47e55. https://doi.org/10.1038/s41587-020-0741-7.

[13] Dane AP, Wowro SJ, Cunningham SC, Alexander IE. Comparison of gene transfer to the murine liver following intraperitoneal and intraportal delivery of hepatotropic AAV pseudo-serotypes. Gene Ther. 2013;20:460e464. https://doi.org/10.1038/gt.2012.67.

[14] Montenegro-Miranda PS, Pa-neda A, ten Bloemendaal L, et al. Adeno-associated viral vector serotype 5 poorly transduces liver in rat models. PLoS One. 2013; 8: e82597. https://doi.org/10.1371/journal.pone.0082597.

[15] Montenegro-Miranda PS, Pichard V, Aubert D, et al. In the rat liver, adenoviral gene transfer efficiency is comparable to AAV. Gene Ther. 2014;21:168e174. https://doi.org/10.1038/gt.2013.69.

[16] de Roos WK, Fallaux FJ, Marinelli AW, et al. Isolated-organ perfusion for local gene delivery: efficient adenovirus-mediated gene transfer into the liver. Gene Ther. 1997;4:55e62. https://doi.org/10.1038/sj.gt.3300362.

[17] Facy O, Doussot A, Zinzindohoué F, Holl S, Rat P, Ortega Deballon P. Isolated hepatic perfusion: principles and results. J Vis Surg. 2014;151:S25eS32. https://doi.org/10.1016/j.jviscsurg.2013.12.006.

[18] Rajeev R, Gamblin TC, Turaga KK. Hepatic perfusion therapy. Surg Clin North Am. 2016;96:357e368. https://doi.org/10.1016/j.suc.2015.12.005.

[19] Gnant MF, Noll LA, Terrill RE, et al. Isolated hepatic perfusion for lapine liver metastases: impact of hyperthermia on permeability of tumor neovasculature. Surgery. 1999;126:890e899.

[20] Reddy SK, Kesmodel SB, Alexander HR. Isolated hepatic perfusion for patients with liver metastases. Ther Adv Med Oncol. 2014;6:180e194. https://doi.org/10.1177/1758834014529175.

[21] Brunetti-Pierri N, Stapleton GE, Law M, et al. Efficient, long-term hepatic gene transfer using clinically relevant HDAd doses by balloon occlusion catheter delivery in nonhuman primates. Mol Ther. 2009;17:327e333. https://doi.org/10.1038/mt.2008.257.

[22] Hodges BL, Taylor KM, Chu Q, et al. Local delivery of a viral vector mitigates neutralization by antiviral antibodies and results in efficient transduction of rabbit liver. Mol Ther. 2005;12:1043e1051. https://doi.org/10.1016/j.ymthe.2005.06.475.

[23] Zabaleta N, Salas D, Paramo M, et al. Improvement of Adeno-associated virus-mediated liver transduction efficacy by regional administration in Macaca fascicularis. Hum Gene Ther Clin Dev. 2017;28:68e73. https://doi.org/10.1089/humc.2016.183.

[24] Paris J, Henderson NC.Liver zonation, revisited. Hepatology. 2022; 76: 1219e1230. https://doi.org/10.1002/hep.32408.

[25] Cunningham RP, Porat-Shliom N. Liver zonation - revisiting old questions with new technologies. Front Physiol. 2021; 12:732929. https://doi.org/10.3389/fphys.2021.732929.

[26] Halpern KB, Shenhav R, Matcovitch-Natan O, et al. Single-cell spatial recon-struction reveals global division of labor in the mammalian liver. Nature. 2017;542:352e356. https://doi.org/10.1038/nature21065.

[27] Cunningham SC, Spinoulas A, Carpenter KH, Wilcken B, Kuchel PW, Alexander IE. AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) Mice. Mol Ther. 2009;17:1340e1346. https://doi.org/10.1038/mt.2009.88.

[28] Fujita S, Eguchi A, Okabe J, et al. Sendai virus-mediated gene delivery into hepatocytes via isolated hepatic perfusion. Biol Pharm Bull. 2006; 29: 1728e1734. https://doi.org/10.1248/bpb.29.1728.

[29] Kinoshita H, Watanabe A, Hisayasu S, Suzuki S, Shimada T. Targeted gene de-livery to selected liver segments via isolated hepatic perfusion. J Surg Res. 2010;160:47e51. https://doi.org/10.1016/j.jss.2010.01.030.

[30] Bell P, Wang L, Gao G, et al. Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates. Mol Genet Metab. 2011;104:395e403. https://doi.org/10.1016/j.ymgme.2011.06.002.

[31] Ginn SL, Christina S, Alexander IE. Genome editing in the human liver: progress and translational considerations. Prog Mol Biol Transl Sci. 2021;182:257e288. https://doi.org/10.1016/bs.pmbts.2021.01.030.

[32] Kok CY, Cunningham SC, Kuchel PW, Alexander IE.Insights into gene therapy for urea cycle defects by mathematical modeling. Hum Gene Ther. 2019; 30: 1385e1394. https://doi.org/10.1089/hum.2019.053.

[33] Barzel A, Paulk NK, Shi Y, et al. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature. 2015;517:360e364. https://doi.org/10.1038/nature13864.

[34] Porro F, Bortolussi G, Barzel A, et al. Promoterless gene targeting without nu-cleases rescues lethality of a Crigler-Najjar syndrome mouse model. EMBO Mol Med. 2017;9:1346e1355. https://doi.org/10.15252/emmm.201707601.

[35] Masuda Y, Yoshizawa K, Ohno Y, Mita A, Shimizu A, Soejima Y. Small-for-size syndrome in liver transplantation: definition, pathophysiology and manage-ment. Hepatobiliary Pancreat Dis Int. 2020;19:334e341. https://doi.org/10.1016/j.hbpd.2020.06.015.

[36] Bartlett JS, Wilcher R, Samulski RJ. Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors. J Virol. 2000; 74: 2777e2785. https://doi.org/10.1128/jvi.74.6.2777-2785.2000.

[37] Ijtsma AJ, van der Hilst CS, de Boer MT, et al. The clinical relevance of the anhepatic phase during liver transplantation. Liver Transpl. 2009; 15: 1050e1055. https://doi.org/10.1002/lt.21791.

[38] Davidoff AM, Gray JT, Ng CY, et al. Comparison of the ability of adeno-associated viral vectors pseudotyped with serotype 2, 5, and 8 capsid pro-teins to mediate efficient transduction of the liver in murine and nonhuman primate models. Mol Ther. 2005;11:875e888. https://doi.org/10.1016/j.ymthe.2004.12.022.

[39] Jacobs F, Wisse E, De Geest B. The role of liver sinusoidal cells in hepatocyte-directed gene transfer. Am J Pathol. 2010;176:14e21. https://doi.org/10.2353/ajpath.2010.090136.

[40] Tanuma Y, Ohata M, Ito T. Electron microscopic studies on the sinusoidal cells in the monkey liver. Arch Histol Jpn. 1983;46:401e426. https://doi.org/10.1679/aohc.46.401.

[41] Vonnahme FJ, Müller O. A scanning electron microscopic study of the liver of the monkey Macaca speciosa. I. Vascular system of the hepatic lobule. Cell Tissue Res. 1981;215:193e205. https://doi.org/10.1007/BF00236259.

[42] Wisse E, Jacobs F, Topal B, Frederik P, De Geest B. The size of endothelial fe-nestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer. Gene Ther. 2008;15:1193e1199. https://doi.org/10.1038/gt.2008.60.