A retrograde approach for liver gene transfer.

The development of hydrodynamic injections was based on the pioneering work by Jon Wolff (1956–2020), who discovered that intracellular delivery of nucleic acids can be achieved by injections of large volumes of naked plasmid DNA solutions1Budker V. Zhang G. Knechtle S. Wolff J.A. Naked DNA delivered intraportally expresses efficiently in hepatocytes.Gene Ther. 1996; 3: 593-598Google Scholar into the portal vein, into the inferior vena cava, or into bile ducts.2Zhang G. Vargo D. Budker V. Armstrong N. Knechtle S. Wolff J.A. Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers.Hum. Gene Ther. 1997; 8: 1763-1772Google Scholar Later, in the late 1990s, two independent groups showed that fast injections of large volumes of solution with naked plasmid DNA into the tail vein of rodents resulted in gene transfer in various organs, particularly the liver.3Liu F. Song Y. Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.Gene Ther. 1999; 6: 1258-1266Google Scholar,4Zhang G. Budker V. Wolff J.A. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.Hum. Gene Ther. 1999; 10: 1735-1737Google Scholar Although clinical application of methods based on the principle of the hydrodynamic injections is limited, this type of injections became very popular for delivery of DNA or RNA in rodents to investigate physiology and/or therapeutic activity of specific genes in the context of a whole animal.In a study published in Molecular Therapy – Methods & Clinical Development, Deplazes and colleagues rejuvenated the hydrodynamic intrabiliary injection originally developed by Wolff and applied it to mouse models of inherited metabolic diseases.5Deplazes S. Schlegel A. Song Z. Allegri G. Rimann N. Scherer T. Willimann M. Opitz L. Cunningham S.C. Alexander I. et al.Intrabiliary infusion of naked DNA vectors targets periportal hepatocytes in mice.Mol. Ther. Methods Clin. Dev. 2022; 27: 352-367Google Scholar Delivery via hydrodynamic retrograde intrabiliary injection (HRII) of DNA minicircles (Figure 1) encoding the OTC gene in spf-ash mice, a model of ornithine transcarbamylase (OTC) deficiency, did not result in sufficient therapeutic efficacy, and further optimization is needed. Nevertheless, the method is clinically attractive because it could be performed through endoscopic retrograde cholangiopancreatography (ERCP), a method that has been in clinical practice for many years and was previously tested by the authors in a large-animal model.6Chan T. Grisch-Chan H.M. Schmierer P. Subotic U. Rimann N. Scherer T. Hetzel U. Bozza M. Harbottle R. Williams J.A. et al.Delivery of non-viral naked DNA vectors to liver in small weaned pigs by hydrodynamic retrograde intrabiliary injection.Mol. Ther. Methods Clin. Dev. 2022; 24: 268-279Google ScholarThe liver is made of hepatocytes operating in repeating units named lobules that are sub-specialized in metabolic processes, a phenomenon termed “liver zonation.” Expression of a given enzyme in its natural layer within the hepatic lobule is expected to be functionally more effective, and more than 50% of liver genes are zonated.7Halpern K.B. Shenhav R. Matcovitch-Natan O. Toth B. Lemze D. Golan M. Massasa E.E. Baydatch S. Landen S. Moor A.E. et al.Single-cell spatial reconstruction reveals global division of labour in the mammalian liver.Nature. 2017; 542: 352-356Google Scholar Because of the metabolic zonation along the porto-central axis, gene therapy should aim at expression of therapeutic proteins within the zone where they are physiologically expressed. In contrast to hydrodynamic tail vein injection resulting in gene transfer to pericentral hepatocytes, HRII resulted in targeting of periportal hepatocytes, the cells normally expressing the urea cycle enzymes (Figure 1).Similarly, adeno-associated virus (AAV) transduction is also not uniform within the hepatic lobule. In mice, for example, AAV8 transduces predominantly hepatocytes near central veins and yields lower transduction levels in hepatocytes in the periportal regions.8Bell P. Wang L. Gao G. Haskins M.E. Tarantal A.F. McCarter R.J. Zhu Y. Yu H. Wilson J.M. Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates.Mol. Genet. Metabol. 2011; 104: 395-403Google Scholar In contrast to mice, in nonhuman primates, the expression pattern from AAV8 vectors is reversed, i.e., transgene expression is higher around portal areas and less intense or absent around central veins.8Bell P. Wang L. Gao G. Haskins M.E. Tarantal A.F. McCarter R.J. Zhu Y. Yu H. Wilson J.M. Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates.Mol. Genet. Metabol. 2011; 104: 395-403Google Scholar A current gene therapy clinical trial for OTC deficiency with AAV is using AAV8 (ClinicalTrials.gov: NCT05345171), and thus, if the pattern of transduction is similar between nonhuman primates and humans, this approach may not provide expression of the OTC in the cells physiologically expressing the enzyme.In mice, other AAV serotypes have been found to transduce preferentially periportal hepatocytes9Cabanes-Creus M. Navarro R.G. Liao S.H.Y. Baltazar G. Drouyer M. Zhu E. Scott S. Luong C. Wilson L.O.W. Alexander I.E. Lisowski L. Single amino acid insertion allows functional transduction of murine hepatocytes with human liver tropic AAV capsids.Mol. Ther. Methods Clin. Dev. 2021; 21: 607-620Google Scholar with an opposite pattern compared with AAV8. However, studies in nonhuman primates are still to be performed to establish the transduction pattern of these vectors within the hepatic lobule. The study by Deplazes and colleagues raises the attractive possibility to use the same route of delivery method used for minicircle DNA also for AAV delivery. The advantages of AAV delivery by HRII include the potential of reducing both the dose of vector and toxicity. The development of hydrodynamic injections was based on the pioneering work by Jon Wolff (1956–2020), who discovered that intracellular delivery of nucleic acids can be achieved by injections of large volumes of naked plasmid DNA solutions1Budker V. Zhang G. Knechtle S. Wolff J.A. Naked DNA delivered intraportally expresses efficiently in hepatocytes.Gene Ther. 1996; 3: 593-598Google Scholar into the portal vein, into the inferior vena cava, or into bile ducts.2Zhang G. Vargo D. Budker V. Armstrong N. Knechtle S. Wolff J.A. Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers.Hum. Gene Ther. 1997; 8: 1763-1772Google Scholar Later, in the late 1990s, two independent groups showed that fast injections of large volumes of solution with naked plasmid DNA into the tail vein of rodents resulted in gene transfer in various organs, particularly the liver.3Liu F. Song Y. Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.Gene Ther. 1999; 6: 1258-1266Google Scholar,4Zhang G. Budker V. Wolff J.A. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.Hum. Gene Ther. 1999; 10: 1735-1737Google Scholar Although clinical application of methods based on the principle of the hydrodynamic injections is limited, this type of injections became very popular for delivery of DNA or RNA in rodents to investigate physiology and/or therapeutic activity of specific genes in the context of a whole animal. In a study published in Molecular Therapy – Methods & Clinical Development, Deplazes and colleagues rejuvenated the hydrodynamic intrabiliary injection originally developed by Wolff and applied it to mouse models of inherited metabolic diseases.5Deplazes S. Schlegel A. Song Z. Allegri G. Rimann N. Scherer T. Willimann M. Opitz L. Cunningham S.C. Alexander I. et al.Intrabiliary infusion of naked DNA vectors targets periportal hepatocytes in mice.Mol. Ther. Methods Clin. Dev. 2022; 27: 352-367Google Scholar Delivery via hydrodynamic retrograde intrabiliary injection (HRII) of DNA minicircles (Figure 1) encoding the OTC gene in spf-ash mice, a model of ornithine transcarbamylase (OTC) deficiency, did not result in sufficient therapeutic efficacy, and further optimization is needed. Nevertheless, the method is clinically attractive because it could be performed through endoscopic retrograde cholangiopancreatography (ERCP), a method that has been in clinical practice for many years and was previously tested by the authors in a large-animal model.6Chan T. Grisch-Chan H.M. Schmierer P. Subotic U. Rimann N. Scherer T. Hetzel U. Bozza M. Harbottle R. Williams J.A. et al.Delivery of non-viral naked DNA vectors to liver in small weaned pigs by hydrodynamic retrograde intrabiliary injection.Mol. Ther. Methods Clin. Dev. 2022; 24: 268-279Google Scholar The liver is made of hepatocytes operating in repeating units named lobules that are sub-specialized in metabolic processes, a phenomenon termed “liver zonation.” Expression of a given enzyme in its natural layer within the hepatic lobule is expected to be functionally more effective, and more than 50% of liver genes are zonated.7Halpern K.B. Shenhav R. Matcovitch-Natan O. Toth B. Lemze D. Golan M. Massasa E.E. Baydatch S. Landen S. Moor A.E. et al.Single-cell spatial reconstruction reveals global division of labour in the mammalian liver.Nature. 2017; 542: 352-356Google Scholar Because of the metabolic zonation along the porto-central axis, gene therapy should aim at expression of therapeutic proteins within the zone where they are physiologically expressed. In contrast to hydrodynamic tail vein injection resulting in gene transfer to pericentral hepatocytes, HRII resulted in targeting of periportal hepatocytes, the cells normally expressing the urea cycle enzymes (Figure 1). Similarly, adeno-associated virus (AAV) transduction is also not uniform within the hepatic lobule. In mice, for example, AAV8 transduces predominantly hepatocytes near central veins and yields lower transduction levels in hepatocytes in the periportal regions.8Bell P. Wang L. Gao G. Haskins M.E. Tarantal A.F. McCarter R.J. Zhu Y. Yu H. Wilson J.M. Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates.Mol. Genet. Metabol. 2011; 104: 395-403Google Scholar In contrast to mice, in nonhuman primates, the expression pattern from AAV8 vectors is reversed, i.e., transgene expression is higher around portal areas and less intense or absent around central veins.8Bell P. Wang L. Gao G. Haskins M.E. Tarantal A.F. McCarter R.J. Zhu Y. Yu H. Wilson J.M. Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates.Mol. Genet. Metabol. 2011; 104: 395-403Google Scholar A current gene therapy clinical trial for OTC deficiency with AAV is using AAV8 (ClinicalTrials.gov: NCT05345171), and thus, if the pattern of transduction is similar between nonhuman primates and humans, this approach may not provide expression of the OTC in the cells physiologically expressing the enzyme. In mice, other AAV serotypes have been found to transduce preferentially periportal hepatocytes9Cabanes-Creus M. Navarro R.G. Liao S.H.Y. Baltazar G. Drouyer M. Zhu E. Scott S. Luong C. Wilson L.O.W. Alexander I.E. Lisowski L. Single amino acid insertion allows functional transduction of murine hepatocytes with human liver tropic AAV capsids.Mol. Ther. Methods Clin. Dev. 2021; 21: 607-620Google Scholar with an opposite pattern compared with AAV8. However, studies in nonhuman primates are still to be performed to establish the transduction pattern of these vectors within the hepatic lobule. The study by Deplazes and colleagues raises the attractive possibility to use the same route of delivery method used for minicircle DNA also for AAV delivery. The advantages of AAV delivery by HRII include the potential of reducing both the dose of vector and toxicity. We thank Fondazione Telethon , Italy, for support (to N.B.-P.). N.B.-P. and P.G. wrote this commentary. P.G. is a co-founder of Bloomsbury Genetic Therapies.