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Serotype survey of AAV gene delivery via subconjunctival injection in mice

Abstract

AAV gene therapy approaches in the posterior eye resulted in the first FDA-approved gene therapy-based drug. However, application of AAV vectorology to the anterior eye has yet to enter even a Phase I trial. Furthermore, the simple and safe subconjunctival injection has been relatively unexplored in regard to AAV vector transduction. To determine the utility of this route for the treatment of various ocular disorders, a survey of gene delivery via natural AAV serotypes was performed and correlated to reported cellular attachment factors. AAV serotypes packaged with a self-complementary reporter were administered via subconjunctival injection to WT mice. Subconjunctival injection of AAV vectors was without incidence; however, vector shedding in tears was noted weeks following administration. AAV transduction was serotype dependent in anterior segment tissues including the eye lid, conjunctiva, and cornea, as well as the periocular tissues including muscle. Transgene product in the cornea was highest for AAV6 and AAV8, however, their corneal restriction was remarkably different; AAV6 appeared restricted to the endothelium layer while AAV8 efficiently transduced the stromal layer. Reported AAV cellular receptors were not well correlated to vector transduction; although, in some cases they were conserved among mouse and human ocular tissues. Subconjunctival administration of particular AAV serotypes may be a simple and safe targeted gene delivery route for ocular surface, muscular, corneal, and optic nerve diseases.

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References

  1. 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;358:2231–9.

    CAS  Article  Google Scholar 

  2. Feuer WJ, Schiffman JC, Davis JL, Porciatti V, Gonzalez P, Koilkonda RD, et al. Gene therapy for Leber hereditary optic neuropathy: initial results. Ophthalmology. 2016;123:558–70.

    Article  Google Scholar 

  3. Chong RS, Su DH, Tsai A, Jiang Y, Htoon HM, Lamoureux EL, et al. Patient acceptance and attitude toward an alternative method of subconjunctival injection for the medical treatment of glaucoma. J Glaucoma. 2013;22:190–4.

    Article  Google Scholar 

  4. Salganik M, Hirsch ML, Samulski RJ. Adeno-associated virus as a mammalian DNA vector. Microbiol Spectr. 2015;3:1–32.

  5. Atchison RW, Casto BC, Hammon WM. Adenovirus-associated defective virus particles. Science. 1965;149:754–6.

    CAS  Article  Google Scholar 

  6. Atchison RW, Casto BC, Hammon WM. Electron microscopy of adenovirus-associated virus (AAV) in cell cultures. Virology. 1966;29:353–7.

    CAS  Article  Google Scholar 

  7. Samulski RJ, Srivastava A, Berns KI, Muzyczka N. Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV. Cell. 1983;33:135–43.

    CAS  Article  Google Scholar 

  8. Srivastava A, Lusby EW, Berns KI. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol. 1983;45:555–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Chaudhary K, Moore H, Tandon A, Gupta S, Khanna R, Mohan RR. Nanotechnology and adeno-associated virus-based decorin gene therapy ameliorates peritoneal fibrosis. Am J Physiol Renal Physiol. 2014;307:F777–82.

    CAS  Article  Google Scholar 

  10. Igarashi T, Miyake K, Suzuki N, Kato K, Takahashi H, Ohara K, et al. New strategy for in vivo transgene expression in corneal epithelial progenitor cells. Curr Eye Res. 2002;24:46–50.

    Article  Google Scholar 

  11. Mohan RR, Schultz GS, Hong JW, Wilson SE. Gene transfer into rabbit keratocytes using AAV and lipid-mediated plasmid DNA vectors with a lamellar flap for stromal access. Exp Eye Res. 2003;76:373–83.

    CAS  Article  Google Scholar 

  12. Mohan RR, Sharma A, Netto MV, Sinha S, Wilson SE. Gene therapy in the cornea. Prog Retin Eye Res. 2005;24:537–59.

    CAS  Article  Google Scholar 

  13. Sharma A, Tovey JC, Ghosh A, Mohan RR. AAV serotype influences gene transfer in corneal stroma in vivo. Exp Eye Res. 2010;91:440–8.

    CAS  Article  Google Scholar 

  14. Mohan RR, Sharma A, Cebulko TC, Tandon A. Vector delivery technique affects gene transfer in the cornea in vivo. Mol Vis. 2010;16:2494–501.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Mohan RR, Tovey JC, Sharma A, Schultz GS, Cowden JW, Tandon A. Targeted decorin gene therapy delivered with adeno-associated virus effectively retards corneal neovascularization in vivo. PLoS ONE. 2011;6:e26432.

    CAS  Article  Google Scholar 

  16. Mohan RR, Tandon A, Sharma A, Cowden JW, Tovey JC. Significant inhibition of corneal scarring in vivo with tissue-selective, targeted AAV5 decorin gene therapy. Invest Ophthalmol Vis Sci. 2011;52:4833–41.

    CAS  Article  Google Scholar 

  17. Mohan RR, Sinha S, Tandon A, Gupta R, Tovey JC, Sharma A. Efficacious and safe tissue-selective controlled gene therapy approaches for the cornea. PLoS ONE. 2011;6:e18771.

    CAS  Article  Google Scholar 

  18. Vance M, Llanga T, Bennett W, Woodard K, Murlidharan G, Chungfat N, et al. AAV gene therapy for MPS1-associated corneal blindness. Sci Rep. 2016;6:22131.

    CAS  Article  Google Scholar 

  19. Hippert C, Ibanes S, Serratrice N, Court F, Malecaze F, Kremer EJ, et al. Corneal transduction by intra-stromal injection of AAV vectors in vivo in the mouse and ex vivo in human explants. PLoS ONE. 2012;7:e35318.

    CAS  Article  Google Scholar 

  20. Tsai ML, Chen SL, Chou PI, Wen LY, Tsai RJ, Tsao YP. Inducible adeno-associated virus vector-delivered transgene expression in corneal endothelium. Invest Ophthalmol Vis Sci. 2002;43:751–7.

    PubMed  Google Scholar 

  21. Bogner B, Boye SL, Min SH, Peterson JJ, Ruan Q, Zhang Z, et al. Capsid mutated adeno-associated virus delivered to the anterior chamber results in efficient transduction of trabecular meshwork in mouse and rat. PLoS ONE. 2015;10:e0128759.

    Article  Google Scholar 

  22. Buie LK, Rasmussen CA, Porterfield EC, Ramgolam VS, Choi VW, Markovic-Plese S, et al. Self-complementary AAV virus (scAAV) safe and long-term gene transfer in the trabecular meshwork of living rats and monkeys. Invest Ophthalmol Vis Sci. 2010;51:236–48.

    Article  Google Scholar 

  23. O’Callaghan J, Crosbie DE, Cassidy PS, Sherwood JM, Flügel-Koch C, Lütjen-Drecoll E, et al. Therapeutic potential of AAV-mediated MMP-3 secretion from corneal endothelium in treating glaucoma. Hum Mol Genet. 2017;26:1230–1246.

    Article  Google Scholar 

  24. Wang L, Xiao R, Andres-Mateos E, Vandenberghe LH. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS ONE. 2017;12:e0182473.

    Article  Google Scholar 

  25. Thomas PB, Samant DM, Selvam S, Wei RH, Wang Y, Stevenson D, et al. Adeno-associated virus-mediated IL-10 gene transfer suppresses lacrimal gland immunopathology in a rabbit model of autoimmune dacryoadenitis. Invest Ophthalmol Vis Sci. 2010;51:5137–44.

    Article  Google Scholar 

  26. Cheng HC, Yeh SI, Tsao YP, Kuo PC. Subconjunctival injection of recombinant AAV-angiostatin ameliorates alkali burn induced corneal angiogenesis. Mol Vis. 2007;13:2344–52.

    PubMed  Google Scholar 

  27. Igarashi T, Miyake K, Asakawa N, Miyake N, Shimada T, Takahashi H. Direct comparison of administration routes for AAV8-mediated ocular gene therapy. Curr Eye Res. 2013;38:569–77.

    CAS  Article  Google Scholar 

  28. Lai LJ, Xiao X, Wu JH. Inhibition of corneal neovascularization with endostatin delivered by adeno-associated viral (AAV) vector in a mouse corneal injury model. J Biomed Sci. 2007;14:313–22.

    CAS  Article  Google Scholar 

  29. Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. New Engl J Med. 2011;365:2357–65.

    CAS  Article  Google Scholar 

  30. Nathwani AC, Nienhuis AW, Davidoff AM. Our journey to successful gene therapy for hemophilia B. Hum Gene Ther. 2014;25:923–6.

    CAS  Article  Google Scholar 

  31. Szybalski W. The 50th anniversary of gene therapy: beginnings and present realities. Gene. 2013;525:151–4.

    CAS  Article  Google Scholar 

  32. Schwartz AE, Rodrigues MM, Brown K, Gaskins R, Hackett J, Thomas G, et al. Corneal opacification in C57BL/6J mice. Cornea. 1982;1:195–204.

    Article  Google Scholar 

  33. Koehn D, Meyer KJ, Syed NA, Anderson MG. Ketamine/xylazine-induced corneal damage in mice. PLoS ONE. 2015;10:e0132804.

    Article  Google Scholar 

  34. Nathwani AC, Reiss UM, Tuddenham EG, Rosales C, Chowdary P, McIntosh J, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. New Engl J Med. 2014;371:1994–2004.

    Article  Google Scholar 

  35. Summerford C, Samulski RJ. Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol. 1998;72:1438–45.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Qiu J, Mizukami H, Brown KE. Adeno-associated virus 2 co-receptors? Nat Med. 1999;5:467–8.

    CAS  Article  Google Scholar 

  37. Weller ML, Amornphimoltham P, Schmidt M, Wilson PA, Gutkind JS, Chiorini JA. Epidermal growth factor receptor is a co-receptor for adeno-associated virus serotype 6. Nat Med. 2010;16:662–4.

    CAS  Article  Google Scholar 

  38. Pillay S, Meyer NL, Puschnik AS, Davulcu O, Diep J, Ishikawa Y, et al. An essential receptor for adeno-associated virus infection. Nature. 2016;530:108–12.

    CAS  Article  Google Scholar 

  39. Akache B, Grimm D, Pandey K, Yant SR, Xu H, Kay MA. The 37/67-kilodalton laminin receptor is a receptor for adeno-associated virus serotypes 8, 2, 3, and 9. J Virol. 2006;80:9831–6.

    CAS  Article  Google Scholar 

  40. Mietzsch M, Broecker F, Reinhardt A, Seeberger PH, Heilbronn R. Differential adeno-associated virus serotype-specific interaction patterns with synthetic heparins and other glycans. J Virol. 2014;88:2991–3003.

    Article  Google Scholar 

  41. Wu Z, Miller E, Agbandje-McKenna M, Samulski RJ. Alpha2,3 andalpha2,6 N-linked sialic acids facilitate efficient binding and transduction by adeno-associated virus types 1 and 6. J Virol. 2006;80:9093–103.

    CAS  Article  Google Scholar 

  42. Kaludov N, Brown KE, Walters RW, Zabner J, Chiorini JA. Adeno-associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity. J Virol. 2001;75:6884–93.

    CAS  Article  Google Scholar 

  43. Rocha EM, Di Pasquale G, Riveros PP, Quinn K, Handelman B, Chiorini JA. Transduction, tropism, and biodistribution of AAV vectors in the lacrimal gland. Invest Ophthalmol Vis Sci. 2011;52:9567–72.

    Article  Google Scholar 

  44. Li SK, Hao J, Liu H, Lee JH. MRI study of subconjunctival and intravitreal injections. J Pharm Sci. 2012;101:2353–63.

    CAS  Article  Google Scholar 

  45. Gaudana R, Ananthula HK, Parenky A, Mitra AK. Ocular drug delivery. AAPS J. 2010;12:348–60.

    CAS  Article  Google Scholar 

  46. Hewitt FC, Li C, Gray SJ, Cockrell S, Washburn M, Samulski RJ. Reducing the risk of adeno-associated virus (AAV) vector mobilization with AAV type 5 vectors. J Virol. 2009;83:3919–29.

    CAS  Article  Google Scholar 

  47. Li C, Goudy K, Hirsch M, Asokan A, Fan Y, Alexander J, et al. Cellular immune response to cryptic epitopes during therapeutic gene transfer. Proc Natl Acad Sci USA. 2009;106:10770–4.

    CAS  Article  Google Scholar 

  48. Li C, Hirsch M, DiPrimio N, Asokan A, Goudy K, Tisch R, et al. Cytotoxic-T-lymphocyte-mediated elimination of target cells transduced with engineered adeno-associated virus type 2 vector in vivo. J Virol. 2009;83:6817–24.

    CAS  Article  Google Scholar 

  49. Bennett J, Ashtari M, Wellman J, Marshall KA, Cyckowski LL, Chung DC, et al. AAV2 gene therapy readministration in three adults with congenital blindness. Sci Transl Med. 2012;4:120ra15.

    Article  Google Scholar 

  50. 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;14:1760–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Amado D, Mingozzi F, Hui D, Bennicelli JL, Wei Z, Chen Y, et al. Safety and efficacy of subretinal readministration of a viral vector in large animals to treat congenital blindness. Sci Transl Med. 2010;2:21ra16.

    Article  Google Scholar 

  52. Gilger BC. Immune relevant models for ocular inflammatory diseases. ILAR J. 2018:1–11.

  53. Hirsch ML, Conatser LM, Smith SM, Salmon JH, Wu J, Buglak NE, et al. AAV vector-meditated expression of HLA-G reduces injury-induced corneal vascularization, immune cell infiltration, and fibrosis. Sci Rep. 2017;7:17840.

    Article  Google Scholar 

  54. Song L, Li X, Jayandharan GR, Wang Y, Aslanidi GV, Ling C, et al. High-efficiency transduction of primary human hematopoietic stem cells and erythroid lineage-restricted expression by optimized AAV6 serotype vectors in vitro and in a murine xenograft model in vivo. PLoS ONE. 2013;8:e58757.

    CAS  Article  Google Scholar 

  55. Song L, Kauss MA, Kopin E, Chandra M, Ul-Hasan T, Miller E, et al. Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo. Cytotherapy. 2013;15:986–98.

    CAS  Article  Google Scholar 

  56. Grieger JC, Choi VW, Samulski RJ. Production and characterization of adeno-associated viral vectors. Nat Protoc. 2006;1:1412–28.

    CAS  Article  Google Scholar 

  57. Llanga T, Nagy N, Conatser L, Dial C, Sutton RB, Hirsch ML. Structure-based designed nano-dysferlin significantly improves dysferlinopathy in BLA/J mice. Mol Ther. 2017;25:2150–2162.

    CAS  Article  Google Scholar 

  58. Ye L, Yu H, Li C, Hirsch ML, Zhang L, Samulski RJ, et al. Adeno-associated virus vector mediated delivery of the HBV genome induces chronic hepatitis B virus infection and liver fibrosis in mice. PLoS ONE. 2015;10:e0130052.

    Article  Google Scholar 

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Acknowledgements

This study was supported by grants from the NIH RO1AI072176-06A1 (MH), RO1AR064369-01A1 (MH), and Education Bureau of Hunan Province 14B112 (LJS). A portion of the imaging was done using the Neuroscience Center Microscopy Core Facility equipment, which is supported by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant U54HD079124. The authors thank the Vector Core at the University of North Carolina for providing the AAV vectors used in this study, the CGIBD Histology Core and histology technician, Carolyn Suitt, for the work of tissue processing and sectioning, the Animal Histopathology and laboratory Medicine Core and Dr. Ling Wang for the clinical services, the Microscopy Core Facility of the Neuroscience Center and Dr. Michelle S. Itano for the valuable technical assistance in confocal imaging, Dr. Hua Mei for reviewing the data, and Jerry Wu for manuscript proofreading.

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Correspondence to Matthew L. Hirsch.

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Matthew Hirsch has several licensed patents not related to this report and has received royalties from Fortress Biotech and Asklepios BioPharmaceutical. Matthew Hirsch is a consultant to Tamid Bio.

Electronic supplementary material

Supplementary Figure Legend

41434_2018_35_MOESM2_ESM.tif

Figure S1:Vector Characterization. (A) Viral genome integrity assessment by alkaline electrophoresis followed by SYBR Gold staining. (B) Vector purity examination by silver staining of AAV capsid proteins.

41434_2018_35_MOESM3_ESM.tif

Figure S2: Safety analysis. (A) Changes in body weight during the experiment; (B) Comparison of activity of ALT in serum obtained pre-, 2 weeks and 8 weeks post-injection. (C) Quantitative evaluation of clinical histopathology scores of H&E stained sections.

41434_2018_35_MOESM4_ESM.jpg

Figure S3: Southern blotting detection of mouse ß-actin in tears. Tear samples collected at 1 week (upper panel) and 4 week (lower panel) post-subconjunctival injection were subjected to two rounds of PCR using mouse ß-actin primer set and detected by mouse ß-actin specific probe via Southern blotting. Viral vector genome plus host cell gDNA was used as positive control template.

41434_2018_35_MOESM5_ESM.jpg

Figure S4: AAV receptor analysis in mouse conjunctiva (A) and retina (B) by immunofluorescence staining. Anti-HS antibody, anti-EGFR antibody, anti-67 KDa Lam R antibody (recognizes both 37KDa Lam R precursor and 67KDa Lam R) and anti-AAVR antibody were used for HSPG, EGFR, 37/67 KDa Lam R and AAVR, respectively. WGA, SNA and MAL I were used for staining of multivalent sialic acid, α2, 6 sialic acid and α2, 3 sialic acid, respectively. NC-1: Negative controls (no primary antibody) for EGFR, 37/67 KDa Lam R and AAVR staining; NC-2: Negative controls (no primary antibody) for HSPG staining; NC-3: Negative controls for sialic acid staining. Scale Bar=20 µm.

41434_2018_35_MOESM6_ESM.jpg

Figure S5: Representative negative control images. Negative controls for WGA staining in Fig. 5 (a, b, c & d). Negative controls for SNA staining in Fig. 5 (e, f, g & h). Negative controls (no primary antibody) for AAVR staining in Fig. 5 (i & k). Negative controls (no primary antibody) for longer exposure time used for the stromal layer of Lam R and AAVR staining (j). Negative controls (no primary antibody) for EGFR and 37/67 KDa Lam R staining in Fig. 5, (l, m, n & o). Negative controls for MAL I staining in Fig. 5 (p, q, r & s).

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Song, L., Llanga, T., Conatser, L.M. et al. Serotype survey of AAV gene delivery via subconjunctival injection in mice. Gene Ther 25, 402–414 (2018). https://doi.org/10.1038/s41434-018-0035-6

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