Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates

Gene Therapy volume 19, pages 999–1009 (2012)Cite this article

Subjects

Abstract

We report on the long-term safety of AAV2.sFlt-1 (a recombinant adeno-associated virus serotype 2 carrying the soluble form of the Flt-1 receptor) injection into the subretinal space of non-human primates. Levels of sFlt-1 protein were significantly higher (P<0.05) in the vitreous of four out of five AAV2.sFlt-1-injected eyes. There was no evidence of damage to the eyes of animals that received subretinal injections of AAV2.sFlt-1; ocular examination showed no anterior chamber flare, normal fundus and electroretinography responses equivalent to those observed before treatment. Notably, immunological analysis demonstrated that gene therapy involving subretinal injection of AAV2.sFlt-1 does not elicit cell-mediated immunity. Biodistribution analysis showed that AAV2.sFlt-1 could be detected only in the eye and not in the other organs tested. These data indicate that gene therapy with subretinal AAV2.sFlt-1 is safe and well tolerated, and therefore promising for the long-term treatment of neovascular diseases of the eye.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Adamis AP, Miller JW, Bernal MT, D'Amico DJ, Folkman J, Yeo TK et al. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol 1994; 118: 445–450.

    Article  CAS  PubMed  Google Scholar 

  2. Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD, Shah ST et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994; 331: 1480–1487.

    Article  CAS  PubMed  Google Scholar 

  3. Campochiaro PA, Choy DF, Do DV, Hafiz G, Shah SM, Nguyen QD et al. Monitoring ocular drug therapy by analysis of aqueous samples. Ophthalmology 2009; 116: 2158–2164.

    Article  PubMed  Google Scholar 

  4. Lai CM, Dunlop SA, May LA, Gorbatov M, Brankov M, Shen WY et al. Generation of transgenic mice with mild and severe retinal neovascularization. Br J Ophthalmol 2005; 89: 911–916.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Lai YK, Shen WY, Brankov M, Lai CM, Constable IJ, Rakoczy PE . Potential long-term inhibition of ocular neovascularization by recombinant adeno-associated virus-mediated secretion gene therapy. Gene Therapy 2002; 9: 804–813.

    Article  CAS  PubMed  Google Scholar 

  6. Okamoto N, Tobe T, Hackett SF, Ozaki H, Vinores MA, LaRochelle W 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 

  7. Ozaki H, Hayashi H, Vinores SA, Moromizato Y, Campochiaro PA, Oshima K . Intravitreal sustained release of VEGF causes retinal neovascularization in rabbits and breakdown of the blood-retinal barrier in rabbits and primates. Exp Eye Res 1997; 64: 505–517.

    Article  CAS  PubMed  Google Scholar 

  8. Aiello LP, Pierce EA, Foley ED, Takagi H, Chen H, Riddle L et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA 1995; 92: 10457–10461.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bainbridge JW, Mistry A, De Alwis M, Paleolog E, Baker A, Thrasher AJ 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 

  10. Krzystolik MG, Afshari MA, Adamis AP, Gaudreault J, Gragoudas ES, Michaud NA et al. Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment. Arch Ophthalmol 2002; 120: 338–346.

    Article  CAS  PubMed  Google Scholar 

  11. Lai CM, Shen WY, Brankov M, Lai YK, Barnett NL, Lee SY et al. Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys. Mol Ther 2005; 12: 659–668.

    Article  CAS  PubMed  Google Scholar 

  12. Lynch SS, Cheng CM . Bevacizumab for neovascular ocular diseases. Ann Pharmacother 2007; 41: 614–625.

    Article  CAS  PubMed  Google Scholar 

  13. Rosenfeld PJ, Brown DM, Heier JS, Boyer DS, Kaiser PK, Chung CY et al. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med 2006; 355: 1419–1431.

    Article  CAS  PubMed  Google Scholar 

  14. Zhou B, Wang B . Pegaptanib for the treatment of age-related macular degeneration. Exp Eye Res 2006; 83: 615–619.

    Article  CAS  PubMed  Google Scholar 

  15. Brown DM, Michels M, Kaiser PK, Heier JS, Sy JP, Ianchulev T . Ranibizumab versus verteporfin photodynamic therapy for neovascular age-related macular degeneration: two-year results of the ANCHOR study. Ophthalmology 2009; 116: 57–65 e5.

    Article  PubMed  Google Scholar 

  16. Regillo CD, Brown DM, Abraham P, Yue H, Ianchulev T, Schneider S et al. Randomized, double-masked, sham-controlled trial of ranibizumab for neovascular age-related macular degeneration: PIER Study year 1. Am J Ophthalmol 2008; 145: 239–248.

    Article  CAS  PubMed  Google Scholar 

  17. Schmidt-Erfurth U, Eldem B, Guymer R, Korobelnik JF, Schlingemann RO, Axer-Siegel R et al. Efficacy and safety of monthly versus quarterly ranibizumab treatment in neovascular age-related macular degeneration The EXCITE Study. Ophthalmology 2010; 118: 831–839.

    Article  PubMed  Google Scholar 

  18. Fintak DR, Shah GK, Blinder KJ, Regillo CD, Pollack J, Heier JS et al. Incidence of endophthalmitis related to intravitreal injection of bevacizumab and ranibizumab. Retina 2008; 28: 1395–1399.

    Article  PubMed  Google Scholar 

  19. Chang LK, Sarraf D . Tears of the retinal pigment epithelium: an old problem in a new era. Retina 2007; 27: 523–534.

    Article  PubMed  Google Scholar 

  20. Lai CM, Estcourt MJ, Wikstrom M, Himbeck RP, Barnett NL, Brankov M et al. rAAV.sFlt-1 gene therapy achieves lasting reversal of retinal neovascularization in the absence of a strong immune response to the viral vector. Invest Ophthalmol Vis Sci 2009; 50: 4279–4287.

    Article  PubMed  Google Scholar 

  21. de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT . The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 1992; 255: 989–991.

    Article  CAS  PubMed  Google Scholar 

  22. Kendall RL, Wang G, Thomas KA . Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem Biophys Res Commun 1996; 226: 324–328.

    Article  CAS  PubMed  Google Scholar 

  23. 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 SRN in age-related macular degeneration. Gene Therapy 2000; 7: 978–985.

    Article  CAS  PubMed  Google Scholar 

  24. Igarashi T, Miyake K, Masuda I, Takahashi H, Shimada T . Adeno-associated vector (type 8)-mediated expression of soluble Flt-1 efficiently inhibits neovascularization in a murine choroidal neovascularization model. Hum Gene Ther 2010; 21: 631–637.

    Article  CAS  PubMed  Google Scholar 

  25. Gehlbach P, Demetriades AM, Yamamoto S, Deering T, Xiao WH, Duh EJ et al. Periocular gene transfer of sFlt-1 suppresses ocular neovascularization and vascular endothelial growth factor-induced breakdown of the blood-retinal barrier. Hum Gene Ther 2003; 14: 129–141.

    Article  CAS  PubMed  Google Scholar 

  26. Rota R, Riccioni T, Zaccarini M, Lamartina S, Gallo AD, Fusco A et al. Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer. J Gene Med 2004; 6: 992–1002.

    Article  CAS  PubMed  Google Scholar 

  27. Ideno J, Mizukami H, Kakehashi A, Saito Y, Okada T, Urabe M et al. Prevention of diabetic retinopathy by intraocular soluble flt-1 gene transfer in a spontaneously diabetic rat model. Int J Mol Med 2007; 19: 75–79.

    CAS  PubMed  Google Scholar 

  28. Lai CM, Brankov M, Zaknich T, Lai YK, Shen WY, Constable IJ 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 

  29. Flotte TR, Brantly ML, Spencer LT, Byrne BJ, Spencer CT, Baker DJ et al. Phase I trial of intramuscular injection of a recombinant adeno-associated virus alpha 1-antitrypsin (rAAV2-CB-hAAT) gene vector to AAT-deficient adults. Hum Gene Ther 2004; 15: 93–128.

    Article  PubMed  Google Scholar 

  30. Moss RB, Rodman D, Spencer LT, Aitken ML, Zeitlin PL, Waltz D et al. Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial. Chest 2004; 125: 509–521.

    Article  PubMed  Google Scholar 

  31. Mease PJ, Hobbs K, Chalmers A, El-Gabalawy H, Bookman A, Keystone E et al. Local delivery of a recombinant adenoassociated vector containing a tumour necrosis factor alpha antagonist gene in inflammatory arthritis: a phase 1 dose-escalation safety and tolerability study. Ann Rheum Dis 2009; 68: 1247–1254.

    Article  CAS  PubMed  Google Scholar 

  32. Kaplitt MG, Feigin A, Tang C, Fitzsimons HL, Mattis P, Lawlor PA et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet 2007; 369: 2097–2105.

    Article  CAS  PubMed  Google Scholar 

  33. McPhee SW, Janson CG, Li C, Samulski RJ, Camp AS, Francis J et al. Immune responses to AAV in a phase I study for Canavan disease. J Gene Med 2006; 8: 577–588.

    Article  CAS  PubMed  Google Scholar 

  34. Manno CS, Chew AJ, Hutchison S, Larson PJ, Herzog RW, Arruda VR et al. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 2003; 101: 2963–2972.

    Article  CAS  PubMed  Google Scholar 

  35. Mingozzi F, Maus MV, Hui DJ, Sabatino DE, Murphy SL, Rasko JE et al. CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat Med 2007; 13: 419–422.

    Article  CAS  PubMed  Google Scholar 

  36. Crystal RG, Sondhi D, Hackett NR, Kaminsky SM, Worgall S, Stieg P et al. Clinical protocol. Administration of a replication-deficient adeno-associated virus gene transfer vector expressing the human CLN2 cDNA to the brain of children with late infantile neuronal ceroid lipofuscinosis. Hum Gene Ther 2004; 15: 1131–1154.

    Article  PubMed  Google Scholar 

  37. Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K et al. Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med 2008; 358: 2231–2239.

    Article  CAS  PubMed  Google Scholar 

  38. Hauswirth W, Aleman TS, Kaushal S, Cideciyan AV, Schwartz SB, Wang L et al. Phase I trial of Leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results. Hum Gene Ther 2008; 19: 979–990.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Maguire AM, Simonelli F, Pierce EA, Pugh Jr EN, Mingozzi F, Bennicelli J et al. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med 2008; 358: 2240–2248.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Frank KM, Hogarth DK, Miller JL, Mandal S, Mease PJ, Samulski RJ et al. Investigation of the cause of death in a gene-therapy trial. N Engl J Med 2009; 361: 161–169.

    Article  CAS  PubMed  Google Scholar 

  41. Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342–347.

    Article  CAS  PubMed  Google Scholar 

  42. Miller JD, van der Most RG, Akondy RS, Glidewell JT, Albott S, Masopust D et al. Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines. Immunity 2008; 28: 710–722.

    Article  CAS  PubMed  Google Scholar 

  43. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H . Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984; 133: 1710–1715.

    CAS  PubMed  Google Scholar 

  44. Chakrabarti LA, Lewin SR, Zhang L, Gettie A, Luckay A, Martin LN et al. Normal T-cell turnover in sooty mangabeys harboring active simian immunodeficiency virus infection. J Virol 2000; 74: 1209–1223.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Pitcher CJ, Hagen SI, Walker JM, Lum R, Mitchell BL, Maino VC et al. Development and homeostasis of T cell memory in rhesus macaque. J Immunol 2002; 168: 29–43.

    Article  CAS  PubMed  Google Scholar 

  46. Johnson CJ, Berglin L, Chrenek MA, Redmond TM, Boatright JH, Nickerson JM . Technical brief: subretinal injection and electroporation into adult mouse eyes. Mol Vis 2008; 14: 2211–2226.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Jacobson SG, Boye SL, Aleman TS, Conlon TJ, Zeiss CJ, Roman AJ et al. Safety in nonhuman primates of ocular AAV2-RPE65, a candidate treatment for blindness in Leber congenital amaurosis. Hum Gene Ther 2006; 17: 845–858.

    Article  CAS  PubMed  Google Scholar 

  48. Provost N, Le Meur G, Weber M, Mendes-Madeira A, Podevin G, Cherel Y et al. Biodistribution of rAAV vectors following intraocular administration: evidence for the presence and persistence of vector DNA in the optic nerve and in the brain. Mol Ther 2005; 11: 275–283.

    Article  CAS  PubMed  Google Scholar 

  49. Dudus L, Anand V, Acland GM, Chen SJ, Wilson JM, Fisher KJ et al. Persistent transgene product in retina, optic nerve and brain after intraocular injection of rAAV. Vision Res 1999; 39: 2545–2553.

    Article  CAS  PubMed  Google Scholar 

  50. Maclachlan TK, Lukason M, Collins M, Munger R, Isenberger E, Rogers C et al. Preclinical safety evaluation of AAV2-sFLT01- a gene therapy for age-related macular degeneration. Mol Ther 2011; 19: 326–334.

    Article  CAS  PubMed  Google Scholar 

  51. Graubert MD, Ortega MA, Kessel B, Mortola JF, Iruela-Arispe ML . Vascular repair after menstruation involves regulation of vascular endothelial growth factor-receptor phosphorylation by sFLT-1. Am J Pathol 2001; 158: 1399–1410.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Barleon B, Reusch P, Totzke F, Herzog C, Keck C, Martiny-Baron G et al. Soluble VEGFR-1 secreted by endothelial cells and monocytes is present in human serum and plasma from healthy donors. Angiogenesis 2001; 4: 143–154.

    Article  CAS  PubMed  Google Scholar 

  53. Wolf M, Hubel CA, Lam C, Sampson M, Ecker JL, Ness RB et al. Preeclampsia and future cardiovascular disease: potential role of altered angiogenesis and insulin resistance. J Clin Endocrinol Metab 2004; 89: 6239–6243.

    Article  CAS  PubMed  Google Scholar 

  54. Chung NA, Lydakis C, Belgore F, Blann AD, Lip GY . Angiogenesis in myocardial infarction. An acute or chronic process? Eur Heart J 2002; 23: 1604–1608.

    Article  CAS  PubMed  Google Scholar 

  55. Lukason M, DuFresne E, Rubin H, Pechan P, Li Q, Kim I et al. Inhibition of choroidal neovascularization in a nonhuman primate model by intravitreal administration of an AAV2 vector expressing a novel anti-VEGF molecule. Mol Ther 2011; 19: 260–265.

    Article  CAS  PubMed  Google Scholar 

  56. Pechan P, Rubin H, Lukason M, Ardinger J, DuFresne E, Hauswirth WW et al. Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization. Gene Therapy 2009; 16: 10–16.

    Article  CAS  PubMed  Google Scholar 

  57. Wiesmann C, Fuh G, Christinger HW, Eigenbrot C, Wells JA, de Vos AM . Crystal structure at 1.7 A resolution of VEGF in complex with domain 2 of the Flt-1 receptor. Cell 1997; 91: 695–704.

    Article  CAS  PubMed  Google Scholar 

  58. Hoffman LM, Maguire AM, Bennett J . Cell-mediated immune response and stability of intraocular transgene expression after adenovirus-mediated delivery. Invest Ophthalmol Vis Sci 1997; 38: 2224–2233.

    CAS  PubMed  Google Scholar 

  59. Li Q, Miller R, Han PY, Pang J, Dinculescu A, Chiodo V et al. Intraocular route of AAV2 vector administration defines humoral immune response and therapeutic potential. Mol Vis 2008; 14: 1760–1769.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Li W, Kong F, Li X, Dai X, Liu X, Zheng Q et al. Gene therapy following subretinal AAV5 vector delivery is not affected by a previous intravitreal AAV5 vector administration in the partner eye. Mol Vis 2009; 15: 267–275.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Shen WY, Garrett KL, Wang CG, Zhang K, Ma ZZ, Constable IJ et al. Preclinical evaluation of a phosphorothioate oligonucleotide in the retina of rhesus monkey. Lab Invest 2002; 82: 167–182.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Juvenile Diabetes Research Foundation (USA), the National Institutes of Health (USA), the National Health and Medical Research Council (Australia) and Westpac grant-in-aid (Australia). MDE holds a NHMRC Principal Research Fellowship.

Author information

Authors and Affiliations

  1. Department of Molecular Ophthalmology, Centre for Ophthalmology and Visual Sciences, The University of Western Australia, Nedlands, Western Australia, Australia

    C-M Lai, R P Himbeck, I J Constable & E P Rakoczy

  2. Centre for Ophthalmology and Visual Sciences, Centre for Experimental Immunology, The University of Western Australia, Nedlands, Western Australia, Australia

    M J Estcourt & M A Degli-Esposti

  3. Singapore National Eye Centre, Singapore, Singapore

    S-Y Lee, I Yew-San Yeo, B K Loh, M W Lee & C-L Ang

  4. Singapore Eye Research Institute, Singapore, Singapore

    C Luu & A Barathi

  5. Department of Experimental Surgery, Singapore General Hospital, Singapore, Singapore

    J Villano

  6. National Research Centre for Asbestos Related Diseases, The University of Western Australia, Nedlands, Western Australia, Australia

    R G van der Most

  7. Joint Vector Core, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    D Dismuke

  8. Department of Pharmacology and Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    R J Samulski

Authors
  1. C-M Lai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M J Estcourt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R P Himbeck
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S-Y Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. I Yew-San Yeo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C Luu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. B K Loh
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M W Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. A Barathi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. J Villano
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. C-L Ang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. R G van der Most
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. I J Constable
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. D Dismuke
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. R J Samulski
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. M A Degli-Esposti
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. E P Rakoczy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to E P Rakoczy.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lai, CM., Estcourt, M., Himbeck, R. et al. Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates. Gene Ther 19, 999–1009 (2012). https://doi.org/10.1038/gt.2011.169

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2011.169

Keywords

This article is cited by

Search

Advanced search

Quick links