Capsid surface shielding of adenovirus vectors with synthetic polymers is an emerging technology to reduce unwanted interactions of the vector particles with cellular and non-cellular host components. While it has been shown that attachment of shielding polymers allows prevention of undesired interactions, it has become evident that a shield which is covalently attached to the vector surface can negatively affect gene transfer efficiency. Reasons are not only a limited receptor-binding ability of the shielded vectors but also a disturbance of intracellular trafficking processes, the latter depending on the interaction of the vector surface with the cellular transport machinery. A solution might be the development of bioresponsive shields that are stably maintained outside the host cell but released upon cell entry to allow for efficient gene delivery to the nucleus. Here we provide a systematic comparison of irreversible versus bioresponsive shields based on synthetic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In addition, the chemical strategy used for generation of the shield allowed for a traceless bioresponsive shielding, i.e., polymers could be released from the vector particles without leaving residual linker residues. Our data demonstrated that only a bioresponsive shield maintained the high gene transfer efficiency of adenovirus vectors both in vitro and in vivo. As an example for bioresponsive HPMA copolymer release, we analyzed the in vivo gene transfer in the liver. We demonstrated that both the copolymer's charge and the mode of shielding (irreversible versus traceless bioresponsive) profoundly affected liver gene transfer and that traceless bioresponsive shielding with positively charged HPMA copolymers mediated FX independent transduction of hepatocytes. In addition, we demonstrated that shielding with HPMA copolymers can mediate a prolonged blood circulation of vector particles in mice. Our results have significant implications for the future design of polymer-shielded Ad and provide a deeper insight into the interaction of shielded adenovirus vector particles with the host after systemic delivery.
References
[1]
Wiley (2013) Journal of gene medicine: Gene therapy clinical trials worldwide. Available: http://www.abedia.com/wiley/. Accessed 2013 March 26.
Carlisle RC, Di Y, Cerny AM, Sonnen AFP, Sim RB, et al. (2009) Human erythrocytes bind and inactivate type 5 adenovirus by presenting coxsackie virus-adenovirus receptor and complement receptor 1. Blood 113: 1909–1918. doi: 10.1182/blood-2008-09-178459
[5]
Wickham TJ, Mathias P, Cheresh DA, Nemerow GR (1993) Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell 73: 309–319. doi: 10.1016/0092-8674(93)90231-e
[6]
Greber UF, Willetts M, Webster P, Helenius A (1993) Stepwise dismantling of adenovirus 2 during entry into cells. Cell 75: 477–486. doi: 10.1016/0092-8674(93)90382-z
[7]
Nakano MY, Boucke K, Suomalainen M, Stidwill RP, Greber UF (2000) The first step of adenovirus type 2 disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol. Journal of Virology 74: 7085–7095. doi: 10.1128/jvi.74.15.7085-7095.2000
[8]
Martin K, Brie A, Saulnier P, Perricaudet M, Yeh P, et al. (2003) Simultaneous CAR- and alpha v integrin-binding ablation fails to reduce ad5 liver tropism. Molecular Therapy: The Journal of the American Society of Gene Therapy 8: 485–494. doi: 10.1016/s1525-0016(03)00182-5
[9]
Kalyuzhniy O, Di Paolo NC, Silvestry M, Hofherr SE, Barry MA, et al. (2008) Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo. Proceedings of the National Academy of Sciences of the United States of America 105: 5483–5488. doi: 10.1073/pnas.0711757105
[10]
Alba R, Bradshaw AC, Parker AL, Bhella D, Waddington SN, et al. (2009) Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer. Blood 114: 965–971. doi: 10.1182/blood-2009-03-208835
[11]
Bradshaw AC, Parker AL, Duffy MR, Coughlan L, van Rooijen N, et al. (2010) Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X. PLoS Pathogens 6: e1001142. doi: 10.1371/journal.ppat.1001142
[12]
Zhang Y, Chirmule N, Gao GP, Qian R, Croyle M, et al. (2001) Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages. Molecular therapy: the journal of the American Society of Gene Therapy 3: 697–707. doi: 10.1006/mthe.2001.0329
[13]
Othman M, Labelle A, Mazzetti I, Elbatarny HS, Lillicrap D (2007) Adenovirus-induced throm-bocytopenia: the role of von willebrand factor and p-selectin in mediating accelerated platelet clearance. Blood 109: 2832–2839. doi: 10.1182/blood-2006-06-032524
[14]
Ganesan LP, Mohanty S, Kim J, Clark KR, Robinson JM, et al. (2011) Rapid and efficient clearance of blood-borne virus by liver sinusoidal endothelium. PLoS Pathogens 7: e1002281. doi: 10.1371/journal.ppat.1002281
[15]
Lyons M, Onion D, Green NK, Aslan K, Rajaratnam R, et al. (2006) Adenovirus type 5 interactions with human blood cells may compromise systemic delivery. Molecular Therapy: The Journal of the American Society of Gene Therapy 14: 118–128. doi: 10.1016/j.ymthe.2006.01.003
[16]
Xu Z, Tian J, Smith JS, Byrnes AP (2008) Clearance of adenovirus by kupffer cells is mediated by scavenger receptors, natural antibodies, and complement. Journal of Virology 82: 11705–11713. doi: 10.1128/jvi.01320-08
[17]
Smith JS, Xu Z, Tian J, Stevenson SC, Byrnes AP (2008) Interaction of systemically delivered adenovirus vectors with kupffer cells in mouse liver. Human Gene Therapy 19: 547–554. doi: 10.1089/hum.2008.004
[18]
Smith JS, Xu Z, Byrnes AP (2008) A quantitative assay for measuring clearance of adenovirus vectors by kupffer cells. Journal of Virological Methods 147: 54–60. doi: 10.1016/j.jviromet.2007.08.009
[19]
Kreppel F, Kochanek S (2008) Modification of adenovirus gene transfer vectors with synthetic polymers: a scientific review and technical guide. Molecular Therapy: The Journal of the American Society of Gene Therapy 16: 16–29. doi: 10.1038/sj.mt.6300321
[20]
Wortmann A, Vhringer S, Engler T, Corjon S, Schirmbeck R, et al. (2008) Fully detargeted polyethylene glycol-coated adenovirus vectors are potent genetic vaccines and escape from pre-existing anti-adenovirus antibodies. Molecular Therapy: The Journal of the American Society of Gene Therapy 16: 154–162. doi: 10.1038/sj.mt.6300306
[21]
Fisher KD, Stallwood Y, Green NK, Ulbrich K, Mautner V, et al. (2001) Polymer-coated adenovirus permits efficient retargeting and evades neutralising antibodies. Gene Therapy 8: 341–348. doi: 10.1038/sj.gt.3301389
[22]
Green NK, Herbert CW, Hale SJ, Hale AB, Mautner V, et al. (2004) Extended plasma circulation time and decreased toxicity of polymer-coated adenovirus. Gene Therapy 11: 1256–1263. doi: 10.1038/sj.gt.3302295
[23]
？ubr V, Kostka L, Selby-Milic T, Fisher K, Ulbrich K, et al. (2009) Coating of adenovirus type 5 with polymers containing quaternary amines prevents binding to blood components. Journal of Controlled Release: Official Journal of the Controlled Release Society 135: 152–158. doi: 10.1016/j.jconrel.2008.12.009
[24]
Espenlaub S, Corjon S, Engler T, Fella C, Ogris M, et al. (2010) Capsomer-specific fluorescent labeling of adenoviral vector particles allows for detailed analysis of intracellular particle trafficking and the performance of bioresponsive bonds for vector capsid modifications. Human Gene Therapy 21: 1155–1167. doi: 10.1089/hum.2009.171
[25]
Wolff JA, Rozema DB (2008) Breaking the bonds: non-viral vectors become chemically dynamic. Mol Ther 16: 8–15. doi: 10.1038/sj.mt.6300326
[26]
Meister A, Anderson ME (1983) Glutathione. Annual review of biochemistry 52: 711–760. doi: 10.1146/annurev.bi.52.070183.003431
[27]
Kosower NS, Kosower EM (1978) The glutathione status of cells. In: GH Bourne JD, Jeon K, editors, International Review of Cytology, Academic Press, volume 54. pp. 109–160.
[28]
Prill JM, Espenlaub S, Samen U, Engler T, Schmidt E, et al. (2010) Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from kupffer cells. Molecular Therapy: The Journal of the American Society of Gene Therapy 19: 83–92. doi: 10.1038/mt.2010.229
[29]
Doronin K, Flatt JW, Di Paolo NC, Khare R, Kalyuzhniy O, et al. (2012) Coagulation factor X activates innate immunity to human species c adenovirus. Science (New York, NY) 338: 795–798. doi: 10.1126/science.1226625
[30]
Holford NH (1986) Clinical pharmacokinetics and pharmacodynamics of warfarin. Understanding the dose-effect relationship. Clinical pharmacokinetics 11: 483–504. doi: 10.2165/00003088-198611060-00005
[31]
Waddington SN, Parker AL, Havenga M, Nicklin SA, Buckley SMK, et al. (2007) Targeting of adenovirus serotype 5 (Ad5) and 5/47 pseudotyped vectors in vivo: fundamental involvement of coagulation factors and redundancy of car binding by ad5. J Virol 81: 9568–9571. doi: 10.1128/jvi.00663-07
[32]
Alba R, Bradshaw AC, Coughlan L, Denby L, McDonald RA, et al. (2010) Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors. Blood 116: 2656–2664. doi: 10.1182/blood-2009-12-260026
[33]
Koizumi N, Yamaguchi T, Kawabata K, Sakurai F, Sasaki T, et al. (2007) Fiber-modified adenovirus vectors decrease liver toxicity through reduced IL-6 production. Journal of Immunology (Baltimore, Md: 1950) 178: 1767–1773. doi: 10.4049/jimmunol.178.3.1767
[34]
Vetter A, Virdi KS, Espenlaub S, R？dl W, Wagner E, et al. (2013) Adenoviral vectors coated with pamam dendrimer conjugates allow car independent virus uptake and targeting to the egf receptor. Mol Pharm 10: 606–618. doi: 10.1021/mp300366f
[35]
Etrych T, ？ubr V, Strohalm J, Sírová M, Ríhová B, et al. (2012) HPMA copolymer-doxorubicin conjugates: The effects of molecular weight and architecture on biodistribution and in vivo activity. J Control Release 164: 346–354. doi: 10.1016/j.jconrel.2012.06.029
[36]
Mann KG (2005) The challenge of regulating anticoagulant drugs: focus on warfarin. Am Heart J 149: S36–S42. doi: 10.1016/j.ahj.2004.10.021
[37]
Xu Z, Qiu Q, Tian J, Smith JS, Conenello GM, et al. (2013) Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement. Nat Med 19: 452–457. doi: 10.1038/nm.3107
[38]
Baker AH, Nicklin SA, Shayakhmetov DM (2013) FX and host defense evasion tactics by adenovirus. Mol Ther 21: 1109–1111. doi: 10.1038/mt.2013.100
[39]
Taylor CG, Potter AJ, Rabinovitch PS (1997) Splenocyte glutathione and CD3-mediated cell proliferation are reduced in mice fed a protein-deficient diet. J Nutr 127: 44–50.
[40]
Khynriam D, Prasad SB (2003) Changes in endogenous tissue glutathione level in relation to murine ascites tumor growth and the anticancer activity of cisplatin. Braz J Med Biol Res 36: 53–63. doi: 10.1590/s0100-879x2003000100008
[41]
Balendiran GK, Dabur R, Fraser D (2004) The role of glutathione in cancer. Cell Biochem Funct 22: 343–352. doi: 10.1002/cbf.1149
[42]
NC3Rs: National Center for the replacement, refinement and reduction in research. Blood sampling in mice: Decision tree for blood sampling. Available: http://www.nc3rs.org.uk/bloodsamplingmic？rosite/page.asp?id=419. Accessed 2013 September 9.
[43]
Diehl KH, Hull R, Morton D, Pfister R, Rabemampianina Y, et al. (2001) A good practice guide to the administration of substances and removal of blood, including routes and volumes. J Appl Toxicol 21: 15–23. doi: 10.1002/jat.727
[44]
Kirby I, Davison E, Beavil AJ, Soh CP, Wickham TJ, et al. (2000) Identification of contact residues and definition of the CAR-binding site of adenovirus type 5 fiber protein. Journal of Virology 74: 2804–2813. doi: 10.1128/jvi.74.6.2804-2813.2000
[45]
Schiedner G, Hertel S, Kochanek S (2000) Efficient transformation of primary human amniocytes by E1 functions of Ad5: generation of new cell lines for adenoviral vector production. Human Gene Therapy 11: 2105–2116. doi: 10.1089/104303400750001417
[46]
Kreppel F, Gackowski J, Schmidt E, Kochanek S (2005) Combined genetic and chemical capsid modifications enable exible and efficient de- and retargeting of adenovirus vectors. Molecular Therapy: The Journal of the American Society of Gene Therapy 12: 107–117. doi: 10.1016/j.ymthe.2005.03.006
[47]
Kreppel F, Biermann V, Kochanek S, Schiedner G (2002) A DNA-based method to assay total and infectious particle contents and helper virus contamination in high-capacity adenoviral vector preparations. Human Gene Therapy 13: 1151–1156. doi: 10.1089/104303402320138934
[48]
Ulbrich K, ？ubr V, Strohalm J, Plocová D, Jelínková M, et al. (2000) Polymeric drugs based on conjugates of synthetic and natural macromolecules. I. synthesis and physico-chemical characterisation. J Control Release 64: 63–79. doi: 10.1016/s0168-3659(99)00141-8
[49]
Reschel T, Konák C, Oupicky D, Seymour LW, Ulbrich K (2002) Physical properties and in vitro transfection efficiency of gene delivery vectors based on complexes of DNA with synthetic polycations. J Control Release 81: 201–217. doi: 10.1016/s0168-3659(02)00045-7
[50]
？ubr V, Ulbrich K (2006) Synthesis and properties of new N-(2-hydroxypropyl) methacrylamide copolymers containing thiazolidine-2-thione reactive groups. Reactive and Functional Polymers 66: 1525–1538. doi: 10.1016/j.reactfunctpolym.2006.05.002
[51]
Gnaccarini C, Peter S, Scheffer U, Vonhoff S, Klussmann S, et al. (2006) Site-specific cleavage of rna by a metal-free artificial nuclease attached to antisense oligonucleotides. J Am Chem Soc 128: 8063–8067. doi: 10.1021/ja061036f
[52]
Carlsson J, Drevin H, Axn R (1978) Protein thiolation and reversible protein-protein conjugation. N-succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem J 173: 723–737.
[53]
Ulbrich K, Etrych T, Chytil P, Jelínková M, Ríhová B (2004) Antibody-targeted polymer-doxorubicin conjugates with pH-controlled activation. J Drug Target 12: 477–489. doi: 10.1080/10611860400011869
