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Nonviral ocular gene transfer

Gene Therapy volume 12, pages 843–851 (2005)Cite this article

Abstract

In this study, we explored the use of electroporation or media that promote lipoplex formation for nonviral gene transfer in the eye. There was no detectable staining for LacZ after subretinal, intravitreous, or periocular injection of a plasmid containing a CMV promoter expression cassette for LacZ, but when plasmid injection in each of the three sites was combined with electroporation, there was efficient transduction. Specific staining for LacZ was seen in retinal pigmented epithelial (RPE) cells after subretinal injection of a plasmid containing a vitelliform macular dystrophy 2 (VMD2) promoter expression cassette, demonstrating that this approach can be used to evaluate purported tissue-specific promoters in vivo. Electroporation with 10 V/mm resulted in strong LacZ staining, but was damaging to photoreceptors; substantial transduction with no evidence of retinal damage was seen using 3.4 V/mm. Staining for LacZ was also seen after subretinal or periocular, but not intravitreous, injection of plasmid DNA in medium containing 40% Lipofectamine2000 (Lf). Injection of 40% Lf into the subretinal space caused damage to photoreceptors, but subretinal injection of plasmid DNA in medium containing 10% NeuroPorter resulted in transduction of RPE cells with no adverse effects on retinal morphology or function as assessed by electroretinograms (ERGs). After either electroporation or lipofection, LacZ staining was detectable for at least 14 days, and could be reinduced by a second procedure. These data suggest that electroporation or lipofection can be used as experimental tools for ocular gene transfer to evaluate tissue-specific promoter fragments or to evaluate the effects of transgene expression in the retina. Also, with additional optimization, nonviral gene transfer may prove to be a valuable approach for the treatment of retinal and choroidal diseases.

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References

  1. Okamoto N et al. Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization. Am J Pathol 1997; 151: 281–291.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Tobe T et al. Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model. Am J Pathol 1998; 153: 1641–1646.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Weng J et al. Insights into the function of rim protein in photoreceptors and etiology of Stargardt's disease from the phenotype in abcr knockout mice. Cell 1999; 98: 13–23.

    Article  CAS  PubMed  Google Scholar 

  4. Seo M-S et al. Photoreceptor-specific expression of PDGF-B results in traction retinal detachment. Am J Pathol 2000; 157: 995–1005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Yamada H et al. Platelet-derived growth factor-A-induced retinal gliosis protects against ischemic retinopathy. Am J Pathol 2000; 156: 477–487.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Yamada H et al. FGF2 decreases hyperoxia-induced cell death in mice. J Am Pathlol 2001; 159: 1113–1120.

    Article  CAS  Google Scholar 

  7. Ando A et al. Nitric oxide is proangiogenic in retina and choroid. J Cell Physiol 2001; 191: 116–124.

    Article  Google Scholar 

  8. Ando A, Yang A, Nambu H, Campochiaro PA . Blockade of nitric-oxide synthase reduces choroidal neovascularization. Mol Pharmacol 2002; 62: 539–544.

    Article  CAS  PubMed  Google Scholar 

  9. Okoye G et al. Increased expression of BDNF preserves retinal function and slows cell death from rhodopsin mutation or oxidative damage. J Neuosci 2003; 23: 4164–4172.

    Article  CAS  Google Scholar 

  10. Bennett J et al. Photoreceptor cell rescue in retinal degeneration (rd) mice by in vivo gene therapy. Nat Med 1996; 2: 649–654.

    Article  CAS  PubMed  Google Scholar 

  11. Mori K et al. Pigment epithelium-derived factor inhibits retinal and choroidal neovascularization. J Cell Physiol 2001; 188: 253–263.

    Article  CAS  PubMed  Google Scholar 

  12. Mori K et al. Inhibition of choroidal neovascularization by intravenous injection of adenoviral vectors expressing secretable endostatin. Am J Pathol 2001; 159: 313–320.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lai C-C et al. Suppression of choroidal neovascularization by adeno-associated virus vector expressing angiostatin. Invest Ophthalmol Vis Sci 2001; 42: 2401–2407.

    CAS  PubMed  Google Scholar 

  14. Acland GM et al. Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 2001; 28: 92–95.

    CAS  PubMed  Google Scholar 

  15. Sanftner LHM, Abel H, Hauswirth WW, Flannery JG . Glial cell line derived neurotrophic factor delays photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. Mol Ther 2001; 4: 622–629.

    Article  Google Scholar 

  16. Bainbridge J et al. Inhibition of retinal neovascularization by gene transfer of soluble VEGF receptor sFlt-1. Gene Therapy 2002; 9: 320–326.

    Article  CAS  PubMed  Google Scholar 

  17. Takahashi K et al. Intraocular expression of endostatin reduces VEGF-induced retinal vascular permeability, neovascularization, and retinal detachment. FASEB J 2003; 17: 896–898 (Epub 2003 Mar 28).

    Article  CAS  PubMed  Google Scholar 

  18. Rasmussen HS et al. Clinical protocol. An open-label, phase I, single administration, dose-escalation study of ADGVPEDF.11D (ADPEDF) in neovascular age-related macular degeneration (AMD). Hum Gene Ther 2001; 12: 2029–2032.

    CAS  PubMed  Google Scholar 

  19. Somiari S et al. Theory and in vivo application of electroporative gene delivery. Mol Ther 2000; 2: 178–187.

    Article  CAS  PubMed  Google Scholar 

  20. Coster HGA . Quantitative analysis of the voltage–current relationships of fixed charge membranes and the associated property of ‘punch-through’. Biophys J 1965; 5: 669–686.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Titomirov AV, Sukharev S, Kistanova E . In vivo electorporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim Biophys Acta 1991; 1088: 131–134.

    Article  CAS  PubMed  Google Scholar 

  22. Zhang L, Li L, Hoffmann GA, Hoffmann RM . Depth-targeted efficient gene delivery and expression in the skin by pulsed electric fields: an approach to gene therapy of skin aging and other diseases. Biochem Biophys Res Commun 1996; 220: 633–636.

    Article  CAS  PubMed  Google Scholar 

  23. Heller R et al. In vivo gene electroinjection and expression in rat liver. FEBS Lett 1996; 389: 225–228.

    Article  CAS  PubMed  Google Scholar 

  24. Oshima Y et al. Targeted gene transfer to corneal endothelium in vivo by electric pulse. Gene Therapy 1998; 5: 1347–1354.

    Article  CAS  PubMed  Google Scholar 

  25. Oshima Y et al. Targeted gene transfer to corneal stroma in vivo by electric pulses. Exp Eye Res 2002; 74: 191–198.

    Article  CAS  PubMed  Google Scholar 

  26. Dezawa M et al. Gene trasfer into retinal ganglion cells by in vivo electroporation: a new approach. Micron 2002; 33: 1–6.

    Article  CAS  PubMed  Google Scholar 

  27. Mo X et al. Rescue of axotomized retinal ganglion cells by BDNF gene electroporation in adult rats. Invest Ophthalmol Vis Sci 2002; 43: 2401–2405.

    PubMed  Google Scholar 

  28. Matsuda T, Cepko CL . Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci USA 2004; 101: 16–22.

    Article  CAS  PubMed  Google Scholar 

  29. Felgner PL et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 1987; 84: 7413–7417.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Brigham KS et al. In vivo transfection of murine lungs with a functioning prokaryotic gene using a liposome vehicle. Am J Med Sci 1989; 298: 278–281.

    Article  CAS  PubMed  Google Scholar 

  31. Ono T, Fujino Y, Tsuchiya T, Tsuda M . Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporated and expressed by brain cells. Neurosci Lett 1990; 117: 259–263.

    Article  CAS  PubMed  Google Scholar 

  32. Zhu N, Liggitt D, Liu Y, Debs R . Systemic gene expression after intravenous DNA delivery into adult mice. Science 1993; 261: 209–211.

    Article  CAS  PubMed  Google Scholar 

  33. Liu F, Song YK, Liu D . Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Therapy 1999; 6: 1258–1266.

    Article  CAS  PubMed  Google Scholar 

  34. Zack DJ et al. Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas. Neuron 1991; 6: 187–199.

    Article  CAS  PubMed  Google Scholar 

  35. Esumi N et al. Analysis of the VMD2 promoter and implication of E-Box binding factors in Its regulation. J Biol Chem 2004; 279: 19064–19073.

    Article  CAS  PubMed  Google Scholar 

  36. Petrukhin K et al. Identification of the gene responsible for Best macular dystrophy. Nat Genet 1998; 19: 241–247.

    Article  CAS  PubMed  Google Scholar 

  37. Cepko CL et al. Lineage analysis with retroviral vectors. Methods Enzymol 2000; 327: 118–145.

    Article  CAS  PubMed  Google Scholar 

  38. Oshima Y et al. Angiopoietin-2 enhances retinal vessel sensitivity to vascular endothelial growth factor. J Cell Physiol 2004; 199: 412–417.

    Article  CAS  PubMed  Google Scholar 

  39. Oshima Y et al. Intraocular gutless adenoviral vectored VEGF stimulates anterior segment but not retinal neovascularization. J Cell Physiol 2004; 199: 399–411.

    Article  CAS  PubMed  Google Scholar 

  40. Jomary C et al. Rescue of photoreceptor function by AAV-mediated gene transfer in a mouse model of inherited retinal degeneration. Gene Therapy 1997; 4: 683–690.

    Article  CAS  PubMed  Google Scholar 

  41. Ali RR et al. Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy. Nat Genet 2000; 25: 306–310.

    Article  CAS  PubMed  Google Scholar 

  42. Vollrath D et al. Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk. Proc Natl Acad Sci USA 2001; 98: 12584–12589.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Lai C-M et al. Inhibition of angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal neovascularization. Hum Gene Ther 2001; 12: 1299–1310.

    Article  CAS  PubMed  Google Scholar 

  44. Cayouette M, Gravel C . Adenovirus-mediated gene transfer of ciliary neurotrophic factor can prevent photoreceptor degeneration in the retinal degeneration (rd) mouse. Hum Gene Ther 1997; 8: 423–430.

    Article  CAS  PubMed  Google Scholar 

  45. Lau D et al. Retinal degeneration is slowed in transgenic rats by AAV-mediated delivery of FGF-2. Invest Ophthalmol Vis Sci 2000; 41: 3622–3633.

    CAS  PubMed  Google Scholar 

  46. Liang F-Q et al. Long-term protection of retinal structure but not function using RAAVCNTF in animal models of retinitis pigmentosa. Mol Ther 2001; 4: 461–472.

    Article  CAS  PubMed  Google Scholar 

  47. Campochiaro PA, Hackett SF . Corneal endothelial cell matrix promotes expression of differentiated features of retinal pigmented epithelial cells: implication of laminin and basic fibroblast growth factor as active components. Exp Eye Res 1993; 57: 539–547.

    Article  CAS  PubMed  Google Scholar 

  48. Gehlbach P et al. Periocular injection of an adenoviral vector encoding pigment epithelium-derived factor inhibits choroidal neovascularization. Gene Therapy 2003; 10: 637–646.

    Article  CAS  PubMed  Google Scholar 

  49. Gehlbach P et al. Periocular gene transfer of sFlt-1 suppresses ocular neovascularization and VEGF-induced breakdown of the blood–retinal barrier. Hum Gene Ther 2003; 14: 129–141.

    Article  CAS  PubMed  Google Scholar 

  50. Mori K et al. Intraocular adenoviral vector-mediated gene transfer is increased in proliferative retinopathies. Invest Ophthalmol Vis Sci 2002; 43: 1610–1615.

    PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by EY05951, EY12609, and core grant P30EY1765 from the NEI; Research to Prevent Blindness Inc. (a Lew R Wasserman Merit Award (PAC) and a senior investigator award (DJZ)); and Dr and Mrs William Lake. PAC is the George S and Dolores Dore Eccles Professor of Ophthalmology. DJZ is the Guerrieri Professor of Genetic Engineering and Molecular Ophthalmology.

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Authors and Affiliations

  1. The Departments of Ophthalmology and Neuroscience, Baltimore, MD, USA

    S Kachi, Y Oshima, N Esumi, M Kachi, B Rogers, D J Zack & P A Campochiaro

  2. The Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    D J Zack

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Kachi, S., Oshima, Y., Esumi, N. et al. Nonviral ocular gene transfer. Gene Ther 12, 843–851 (2005). https://doi.org/10.1038/sj.gt.3302475

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