The AAPS Journal

, Volume 6, Issue 3, pp 72–80

Neural retina limits the nonviral gene transfer to retinal pigment epithelium in an in vitro bovine eye model

  • Leena Pitkänen
  • Jukka Pelkonen
  • Marika Ruponen
  • Seppo Rönkkö
  • Arto Urtti


We investigated the permeation of liposomal and polymeric gene delivery systems through neural retina into retinal pigment epithelium (RPE) and determined the roles of various factors in permeation and subsequent uptake of the delivery systems by RPE. Anterior parts and vitreous of fresh bovine eyes were removed. Retina was left intact or peeled away. Complexes of ethidium monoazide (EMA)-labeled plasmid DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted on the retina or RPE. Two hours later the neural retina was removed, if present, and the RPE cells were detached. Contaminants were removed by sucrose centrifugation, and the RPE cells were analyzed for DNA uptake by flow cytometry. Cellular uptake of FITC-dextrans (molecular weight [mw] 20 000, 500 000 and 2 000 000), FITC-poly-L-lysine (mw 20 000), FITC-labeled oligonucleotide (15-mer), and naked EMA-labeled plasmid DNA was determined after pipetting the solutions on the RPE or neural retina. Location of the fluorescent materials in the retina was visualized with fluorescence microscopy. Neural retina decreased the cellular uptake of DNA complexes by an order of magnitude, the uptake of FITC-dextrans slightly, whereas delivery of polycationic FITC-poly-L-lysine to RPE was almost completely inhibibited. Neural retina decreased the cellular uptake of FITC-oligonucleotides, while the uptake of uncomplexed plasmid was always negligible. conclusions from FACS and fluorescence microscopy were similar: delivery of polymeric and liposomal DNA complexes into RPE are limited by the neural retina. This is due to the size and positive charge of the complexes.


gene delivery intravitreal retina liposome polymer 


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  1. 1.
    Smelser GK, Ishikawa T, Pei YF. Electronmicroscopic studies of intraretinal spaces diffusion of particulate materials. In: Rohen JW, ed. Structure of the eye. II Symp., Stuttgart: Schattauer-Verlaug, 1965;109–120.Google Scholar
  2. 2.
    Marmor MF, Negi A, Maurice DM. Kinetics of macromolecules injected into the subretinal space.Exp Eye Res. 1985;40:687–696.CrossRefPubMedGoogle Scholar
  3. 3.
    Kamei M, Misono K, Lewis H. A study of the ability of tissue plasminogen activator to diffuse into the subretinal space after intravitreal injection in rabbits.Am J Ophthalmol. 1999;128:739–746.CrossRefPubMedGoogle Scholar
  4. 4.
    Hageman GS, Johnson LV. Chondroitin 6-sulfate glycosaminoglycan is a major constituent of primate cone photoreceptor matrix sheaths.Curr Eye Res. 1987;6:639–646.CrossRefPubMedGoogle Scholar
  5. 5.
    Cayouette M, Behn D, Sendtner M, Lachapelle P, Gravel C. Intraocular gene transfer of ciliary neurotrophic factor prevents death and increases responsivenses of rod photoreceptors in the retinal degeneration slow mouse.J Neurosci. 1998;18:9282–9293.PubMedGoogle Scholar
  6. 6.
    Akimoto M, Miyatake S, Kogishi J, et al. Adenovirally expressed basic fibroblast growth factor rescues photoreceptor cells in RCS rats.Invest Ophthalmol Vis Sci. 1999;40:273–279.PubMedGoogle Scholar
  7. 7.
    Bennett J, Zeng Y, Bajwa R, Klatt L, Li Y, Maguire AM. Adenovirus-mediated delivery of rhodopsin-promoted bcl-2 results in a delay in photoreceptor cell death in the rd/rd mouse.Gene Ther. 1998;5:1156–1164.CrossRefPubMedGoogle Scholar
  8. 8.
    Honda M, Sakamoto T, Ishibashi T, Inomata H, Ueno H. Experimental subretinal neovascularization is inhibited by adenovirus-mediated soluble VEGF/flt-1 receptor gene transfection: a role of VEGF and possible treatment for SR in age-related macular degeneration.Gene Ther. 2000;7:978–985.CrossRefPubMedGoogle Scholar
  9. 9.
    Hauswirth WW, Beaufrere L. Ocular gene therapy: Quo vadis?Invest Ophthalmol Vis Sci. 2000;41:2821–2826.PubMedGoogle Scholar
  10. 10.
    Lewin AS, Drenser KA, Hauswirth WW, et al. Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.Nat Med. 1998;4:967–971.CrossRefPubMedGoogle Scholar
  11. 11.
    Miyoshi H, Takahashi M, Gage F, Verma I. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector.Proc Natl Acad Sci USA. 1997;94:10319–10323.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Anglade E, Csaky K. Recombinant adenovirus-mediated gene transfer into the adult rat retina.Curr Eye Res. 1998;17:316–321.CrossRefPubMedGoogle Scholar
  13. 13.
    Li T, Adamian M, Roof D, et al. In vivo transfer of a reporter gene to the retina mediated by an adenoviral vector.Invest Ophthalmol Vis Sci. 1994;35:2543–2549.PubMedGoogle Scholar
  14. 14.
    Rolling F, Shen WY, Tabarias H, et al. Evaluation of adeno-associated virus-mediated gene transfer into the rat retina by clinical fluorescence photography.Hum Gene Ther. 1999;10:641–648.CrossRefPubMedGoogle Scholar
  15. 15.
    Spencer B, Agarwala S, Miskulin M, Smith M, Brandt CR. Herpes simplex virus-mediated gene delivery to the rodent visual system.Invest Ophthalmol Vis Sci. 2000;41:1392–1401.PubMedGoogle Scholar
  16. 16.
    Galileo DS, Hunter K, Smith SB. Stable and efficient gene transfer into the mutant retinal pigment epithelial cells of the Mitf(vit) mouse using a lentiviral vector.Curr Eye Res. 1999;18:135–142.CrossRefPubMedGoogle Scholar
  17. 17.
    Ray J, Wolfe JH Aguirre GD, Haskins ME. Retroviral cDNA transfer to the RPE: stable expression and modification of metabolism.Invest Ophthalmol Vis Sci. 1998;39:1658–1666.PubMedGoogle Scholar
  18. 18.
    Lai YK, Rakoczy P, Constable I, Rolling F. Adeno associated virus-mediated gene transfer into human retinal pigment epithelium cells.Aust NZ J Ophthalmol. 1998;26:77–79.CrossRefGoogle Scholar
  19. 19.
    da Cruz L, Robertson T, Hall MO, Constable IJ, Rakoczy PE. Cell polarity, phagocytosis and viral gene transfer in cultured human retinal pigment epithelial cells.Curr Eye Res. 1998;17:668–672.CrossRefPubMedGoogle Scholar
  20. 20.
    Haeseleer F, Imanishi Y, Saperstein D, Palczewski K. Gene transfer mediated by recombinant baculovirus into mouse eye.Invest Ophthalmol Vis Sci. 2001;42:3294–3300.PubMedPubMedCentralGoogle Scholar
  21. 21.
    Urtti A, Polansky J, Lui GM, Szoka F. Gene delivery and expression in human retinal pigment epithelial cells: effects of synthetic carriers, serum, extracellular matrix and viral promoters.J Drug Target. 2000;7:413–421.CrossRefPubMedGoogle Scholar
  22. 22.
    Abul-Hassan K, Walmsley R, Boulton M. Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells.Curr Eye Res. 2000;20:361–366.CrossRefPubMedGoogle Scholar
  23. 23.
    Pitkänen L, Ruponen M, Nieminen J, Urtti A. Vitreous is a barrier in non viral gene transfer by cationic lipids and polymers.Pharm Res. 2003;20:576–583.CrossRefPubMedGoogle Scholar
  24. 24.
    Boussif O, Lezoualch F, Zanta MD, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyl-eneimine.Proc Natl Acad Sci USA. 1995;92:7297–7301.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Mosser DD, Caron AW, Bourget L, Jolicoeur P, Massie B. Use of a dicistronic expression cassette encoding the green fluorescent protein for the screening and selection of cells expressing inducible gene products.Biotechniques. 1997;22:150–161.PubMedGoogle Scholar
  26. 26.
    Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barriers to gene transfer by a cationic lipid.J Biol Chem. 1995;270:18997–19007.CrossRefPubMedGoogle Scholar
  27. 27.
    Ruponen M, Rönkkö S, Honkakoski P, Pelkonen J, Urtti A. Extracellular glycosaminoglycans modify cellular trafficing of lipoplexes and polyplexes.J Biol Chem. 2001;276:33875–33880.CrossRefPubMedGoogle Scholar
  28. 28.
    McGregor GR, Caskey CT. Construction of plasmids that express E. coli beta- galactosidase in mammalian cells.Nucleic Acids Res. 1989;17:2365.CrossRefGoogle Scholar
  29. 29.
    Feeney-Burns L, Berman E. Isolation of retinal pigment epithelium.Methods Enzymol. 1982;81:95–110.CrossRefPubMedGoogle Scholar
  30. 30.
    Hyvönen Z, Plotniece A, Reine I, Checkavichus B, Duburs G, Urtti A. Novel cationic amphiphilic 1,4-dihydropyridine derivatives for DNA delivery.Biochim Biophys Acta. 2000;1509:451–466.CrossRefPubMedGoogle Scholar
  31. 31.
    Jääskeläinen I, Peltola S, Honkakoski P, Mönkkönen J, Urtti A. A lipid carrier with a membrane active component and a small complex size are required for efficient cellular delivery of anti-sense phosphorothioate oligonucleotides.Eur J Pharm Sci. 2000;10:187–193.CrossRefPubMedGoogle Scholar
  32. 32.
    Hollyfield JG. Hyaluronan and the functional organization of the interphotoreceptor matrix.Invest Ophthalmol Vis Sci. 1999;40:2767–2769.PubMedGoogle Scholar
  33. 33.
    Ruponen M, Ylä-Herttuala S, Urtti A. Interactions of polymeric and liposomal gene delivery systems with extracellular glycosaminoglycans: physicochemical and transfection studies.Biochim Biophys Acta. 1999;1415:331–341.CrossRefPubMedGoogle Scholar
  34. 34.
    Russell SR, Shepherd JD, Hageman GS. Distribution of glycoconjugates in the human retinal internal limiting membrane.Invest Ophthalmol Vis Sci. 1991;32:1986–1995.PubMedGoogle Scholar
  35. 35.
    Heegaard S, Jensen OA, Prause JU. Structure and composition of the inner limiting membrane of the retina. SEM on frozen resin-cracked and enzyme-digested retinas of Macaca mulatta.Graefes Arch Clin Exp Ophthalmol. 1986;224:355–360.CrossRefPubMedGoogle Scholar
  36. 36.
    Chai L, Morris JE. Distribution of heparan sulfate proteoglycans in embryonic chicken neural retina and isolated inner limiting membrane.Curr Eye Res. 1994;13:669–677.CrossRefPubMedGoogle Scholar
  37. 37.
    Azad RF, Driver VB, Tanaka K, Crooke RM, Anderson KP. Antiviral activity of a phosphorothioate oligonucleotide complementary to RNA of the human cytomegalovirus major immediate-early region.Antimicrob Agents Chemother. 1993;37:1945–1954.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Jabs DA, Griffiths PD. Fomivirsen for the treatment of cytomegalovirus retinitis.Am J Ophthalmol. 2002;133:552–556.CrossRefPubMedGoogle Scholar
  39. 39.
    Leeds JM, Henry SP, Truong L, Zutshi A, Levin AA, Kornbrust D. Pharmacokinetics of a potential human cytomegalovirus therapeutic, a phosphorothioate oligonucleotide, after intravitreal injection in the rabbit.Drug Metab Dispos. 1997;25:921–926.PubMedGoogle Scholar
  40. 40.
    Leeds JM, Henry SP, Bistner S, Scherrill S, Williams K, Levin AA. Pharmacokinetics of an antisense oligonucleotide injected intravitreally in monkeys.Drug Metab Dispos. 1998;26:670–675.PubMedGoogle Scholar
  41. 41.
    Rakoczy PE, Lai MC, Watson M, Seydel U, Constable I. Targeted delivery of an antisense oligonucleotide in the retina: uptake, distribution, stability, and effect.Antisense Nucleic Acid Drug Dev. 1996;6:207–213.CrossRefPubMedGoogle Scholar
  42. 42.
    Bennett CF. Antisense oligonucleotides: is the glass half full or half empty?Biochem Pharmacol. 1998;55:9–19.CrossRefPubMedGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2004

Authors and Affiliations

  • Leena Pitkänen
    • 1
    • 2
  • Jukka Pelkonen
    • 3
    • 4
  • Marika Ruponen
    • 1
  • Seppo Rönkkö
    • 1
  • Arto Urtti
    • 1
  1. 1.Department of PharmaceuticsUniversity of KuopioKuopioFinland
  2. 2.Department of OphthalmologyKuopio University HospitalKuopioFinland
  3. 3.Department of Clinical MicrobiologyUniversity of KuopioKuopioFinland
  4. 4.Department of Clinical MicrobiologyKuopio University HospitalKuopioFinland

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