FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Vet Ophthalmol / Gene delivery in the equine cornea: a novel therapeutic stra...
Veterinary ophthalmology2010; 13(5); 301-306; doi: 10.1111/j.1463-5224.2010.00813.x

Gene delivery in the equine cornea: a novel therapeutic strategy.

Abstract: To determine if hybrid adeno-associated virus serotype 2/5 (AAV5) vector can effectively deliver foreign genes into the equine cornea without causing adverse side effects. The aims of this study were to: (i) evaluate efficacy of AAV5 to deliver therapeutic genes into equine corneal fibroblasts (ECFs) using enhanced green fluorescent protein (EGFP) marker gene, and (ii) establish the safety of AAV5 vector for equine corneal gene therapy. Methods: Primary ECF cultures were harvested from healthy donor equine corneas. Cultures were maintained at 37°C in humidified atmosphere with 5% CO(2). Methods: AAV5 vector expressing EGFP under control of hybrid cytomegalovirus + chicken β-actin promoter was applied topically to ECF. Expression of delivered EGFP gene in ECF was quantified using fluorescent microscopy. Using fluorescent staining, the total number of cells and transduction efficiency of tested AAV vector was determined. Phase contrast microscopy, trypan blue and TUNEL assays were used to determine toxicity and safety of AAV5 for ECFs. Results: Topical AAV5 application successfully transduced significant numbers of ECFs. Transduction efficiency was 13.1%. Tested AAV5 vector did not cause phenotype change or significant cell death and cell viability was maintained. Conclusions: Tested AAV5 vector is effective and safe for gene therapy in ECFs in vitro.
Get Full Text
Publication Date: 2010-09-16 PubMed ID: 20840107PubMed Central: PMC3711113DOI: 10.1111/j.1463-5224.2010.00813.xGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • N.I.H.
  • Extramural
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research explores the possibility of using a specific viral vector to inject genes into the cells of a horse’s cornea. The researchers found that their method was safe and successfully delivered the intended genes without damaging the cells they were targeting.

Research Objectives

The research had two primary objectives:

  • To assess the effectiveness of a hybrid adeno-associated virus serotype 2/5 (AAV5) in delivering therapeutic genes into the corneal fibroblasts of the horse, they used the enhanced Green Fluorescent Protein (EGFP) as a marker gene.
  • To establish the safety of using the AAV5 vector for gene therapy in horse corneas.

Methodology

  • The researchers harvested cultures from primary equine corneal fibroblasts (ECFs), which are cells derived from the healthy corneas of horses.
  • These cultures were maintained at a temperature of 37°C in a humid atmosphere with 5% carbon dioxide.
  • They applied the AAV5 vector, which was designed to express EGFP under the control of a hybrid cytomegalovirus and chicken β-actin promoter, topically to the ECFs.
  • They quantified the expression of the EGFP gene that was delivered into the ECFs using fluorescent microscopy.

Evaluating Effectiveness and Safety

They also implemented measures to assess the effectiveness and safety of the AAV5 vector:

  • The total number of cells and the efficiency of the virus vector in transducing, or introducing, genes into the ECFs were determined using fluorescent staining.
  • The researchers made use of phase-contrast microscopy, trypan blue, and TUNEL assays as tools to evaluate the toxicity and safety of the AAV5 vector on the ECFs.

Results

  • The topical application of AAV5 successfully transduced a significant number of ECFs, with a transduction efficiency of 13.1%.
  • The AAV5 vector did not cause any notable change in cell phenotype or significant cell death, indicating that it is safe for use on the cells.
  • The cells’ viability was maintained, further supporting the safety of using AAV5 for gene therapy.

Conclusion

The results of this study indicated that the AAV5 vector evaluated is both effective and safe for use in gene therapy on equine corneal fibroblasts in vitro. This opens opportunities for further research and development in using gene therapy for treating corneal disorders in horses.

Cite This Article

APA
Buss DG, Giuliano E, Sharma A, Mohan RR. (2010). Gene delivery in the equine cornea: a novel therapeutic strategy. Vet Ophthalmol, 13(5), 301-306. https://doi.org/10.1111/j.1463-5224.2010.00813.x

Publication

Veterinary ophthalmology
ISSN: 1463-5224
NlmUniqueID: 100887377
Country: England
Language: English
Volume: 13
Issue: 5
Pages: 301-306

Researcher Affiliations

Buss, Dylan G
  • Harry S. Truman Memorial Veterans Hospital, Columbia, Missouri, USA.
Giuliano, Elizabeth
    Sharma, Ajay
      Mohan, Rajiv R

        MeSH Terms

        • Animals
        • Cornea
        • Dependovirus
        • Genetic Therapy / methods
        • Genetic Therapy / veterinary
        • Genetic Vectors
        • Green Fluorescent Proteins
        • Horses
        • Tissue Culture Techniques

        Grant Funding

        • R01 EY017294-03 / NEI NIH HHS
        • R01 EY017294 / NEI NIH HHS
        • R01-EY-017294 / NEI NIH HHS
        • R01 EY017294-03S2 / NEI NIH HHS
        • I01 BX000357 / BLRD VA

        References

        This article includes 32 references
        1. Mohan RR, Schultz GS, Hong JW, Mohan RR, 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 Mar;76(3):373-83.
          pubmed: 12573666doi: 10.1016/s0014-4835(02)00275-0google scholar: lookup
        2. Mohan RR, Sharma A, Netto MV, Sinha S, Wilson SE. Gene therapy in the cornea.. Prog Retin Eye Res 2005 Sep;24(5):537-59.
          pubmed: 15955719doi: 10.1016/j.preteyeres.2005.04.001google scholar: lookup
        3. Verma IM, Weitzman MD. Gene therapy: twenty-first century medicine.. Annu Rev Biochem 2005;74:711-38.
          pubmed: 15952901doi: 10.1146/annurev.biochem.74.050304.091637google scholar: lookup
        4. Monahan PE, Samulski RJ. Adeno-associated virus vectors for gene therapy: more pros than cons?. Mol Med Today 2000 Nov;6(11):433-40.
          pubmed: 11074369doi: 10.1016/s1357-4310(00)01810-4google scholar: lookup
        5. Daya S, Berns KI. Gene therapy using adeno-associated virus vectors.. Clin Microbiol Rev 2008 Oct;21(4):583-93.
          pmc: PMC2570152pubmed: 18854481doi: 10.1128/cmr.00008-08google scholar: lookup
        6. Michau TM, Schwabenton B, Davidson MG, Gilger BC. Superficial, nonhealing corneal ulcers in horses: 23 cases (1989-2003).. Vet Ophthalmol 2003 Dec;6(4):291-7.
          pubmed: 14641825doi: 10.1111/j.1463-5224.2003.00309.xgoogle scholar: lookup
        7. Brooks DE. Equine Ophthalmology in Veterinary ophthalmology. 4. Ames, Iowa: Blackwell Pub; 2007.
        8. Haber M, Cao Z, Panjwani N, Bedenice D, Li WW, Provost PJ. Effects of growth factors (EGF, PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing.. Vet Ophthalmol 2003 Sep;6(3):211-7.
          pubmed: 12950652doi: 10.1046/j.1463-5224.2003.00296.xgoogle scholar: lookup
        9. Nasisse MP, Nelms S. Equine ulcerative keratitis.. Vet Clin North Am Equine Pract 1992 Dec;8(3):537-55.
          pubmed: 1458328doi: 10.1016/s0749-0739(17)30440-6google scholar: lookup
        10. Monahan PE, Samulski RJ. AAV vectors: is clinical success on the horizon?. Gene Ther 2000 Jan;7(1):24-30.
          pubmed: 10680012doi: 10.1038/sj.gt.3301109google scholar: lookup
        11. Lebherz C, Maguire A, Tang W, Bennett J, Wilson JM. Novel AAV serotypes for improved ocular gene transfer.. J Gene Med 2008 Apr;10(4):375-82.
          pmc: PMC2842078pubmed: 18278824doi: 10.1002/jgm.1126google scholar: lookup
        12. Royo NC, Vandenberghe LH, Ma JY, Hauspurg A, Yu L, Maronski M, Johnston J, Dichter MA, Wilson JM, Watson DJ. Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity.. Brain Res 2008 Jan 23;1190:15-22.
          pmc: PMC2367141pubmed: 18054899doi: 10.1016/j.brainres.2007.11.015google scholar: lookup
        13. Saika S, Yamanaka O, Sumioka T, Miyamoto T, Miyazaki K, Okada Y, Kitano A, Shirai K, Tanaka S, Ikeda K. Fibrotic disorders in the eye: targets of gene therapy.. Prog Retin Eye Res 2008 Mar;27(2):177-96.
          pubmed: 18243038doi: 10.1016/j.preteyeres.2007.12.002google scholar: lookup
        14. Alexander JJ, Hauswirth WW. Adeno-associated viral vectors and the retina.. Adv Exp Med Biol 2008;613:121-8.
          pubmed: 18188936doi: 10.1007/978-0-387-74904-4_13google scholar: lookup
        15. Surace EM, Auricchio A. Versatility of AAV vectors for retinal gene transfer.. Vision Res 2008 Feb;48(3):353-9.
          pubmed: 17923143doi: 10.1016/j.visres.2007.07.027google scholar: lookup
        16. Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K, Viswanathan A, Holder GE, Stockman A, Tyler N, Petersen-Jones S, Bhattacharya SS, Thrasher AJ, Fitzke FW, Carter BJ, Rubin GS, Moore AT, Ali RR. Effect of gene therapy on visual function in Leber's congenital amaurosis.. N Engl J Med 2008 May 22;358(21):2231-9.
          pubmed: 18441371doi: 10.1056/nejmoa0802268google scholar: lookup
        17. Maguire AM, Simonelli F, Pierce EA, Pugh EN Jr, Mingozzi F, Bennicelli J, Banfi S, Marshall KA, Testa F, Surace EM, Rossi S, Lyubarsky A, Arruda VR, Konkle B, Stone E, Sun J, Jacobs J, Dell'Osso L, Hertle R, Ma JX, Redmond TM, Zhu X, Hauck B, Zelenaia O, Shindler KS, Maguire MG, Wright JF, Volpe NJ, McDonnell JW, Auricchio A, High KA, Bennett J. Safety and efficacy of gene transfer for Leber's congenital amaurosis.. N Engl J Med 2008 May 22;358(21):2240-8.
          pmc: PMC2829748pubmed: 18441370doi: 10.1056/nejmoa0802315google scholar: lookup
        18. Auricchio A, Rolling F. Adeno-associated viral vectors for retinal gene transfer and treatment of retinal diseases.. Curr Gene Ther 2005 Jun;5(3):339-48.
          pubmed: 15975011doi: 10.2174/1566523054065020google scholar: lookup
        19. Surace EM, Domenici L, Cortese K, Cotugno G, Di Vicino U, Venturi C, Cellerino A, Marigo V, Tacchetti C, Ballabio A, Auricchio A. Amelioration of both functional and morphological abnormalities in the retina of a mouse model of ocular albinism following AAV-mediated gene transfer.. Mol Ther 2005 Oct;12(4):652-8.
          pubmed: 16023414doi: 10.1016/j.ymthe.2005.06.001google scholar: lookup
        20. Chadderton N, Millington-Ward S, Palfi A, O'Reilly M, Tuohy G, Humphries MM, Li T, Humphries P, Kenna PF, Farrar GJ. Improved retinal function in a mouse model of dominant retinitis pigmentosa following AAV-delivered gene therapy.. Mol Ther 2009 Apr;17(4):593-9.
          pmc: PMC2835099pubmed: 19174761doi: 10.1038/mt.2008.301google scholar: lookup
        21. Van Vliet KM, Blouin V, Brument N, Agbandje-McKenna M, Snyder RO. The role of the adeno-associated virus capsid in gene transfer.. Methods Mol Biol 2008;437:51-91.
          pmc: PMC7120696pubmed: 18369962doi: 10.1007/978-1-59745-210-6_2google scholar: lookup
        22. Ghosh A, Yue Y, Duan D. Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle.. J Gene Med 2006 Mar;8(3):298-305.
          pmc: PMC2581716pubmed: 16385549doi: 10.1002/jgm.835google scholar: lookup
        23. Limberis MP, Vandenberghe LH, Zhang L, Pickles RJ, Wilson JM. Transduction efficiencies of novel AAV vectors in mouse airway epithelium in vivo and human ciliated airway epithelium in vitro.. Mol Ther 2009 Feb;17(2):294-301.
          pmc: PMC2835069pubmed: 19066597doi: 10.1038/mt.2008.261google scholar: lookup
        24. Polyak S, Mah C, Porvasnik S, Herlihy JD, Campbell-Thompson M, Byrne BJ, Valentine JF. Gene delivery to intestinal epithelial cells in vitro and in vivo with recombinant adeno-associated virus types 1, 2 and 5.. Dig Dis Sci 2008 May;53(5):1261-70.
          pmc: PMC3896329pubmed: 17934813doi: 10.1007/s10620-007-9991-1google scholar: lookup
        25. Buss DG, Giuliano EA, Sharma A, Mohan RR. Isolation and cultivation of equine corneal keratocytes, fibroblasts and myofibroblasts.. Vet Ophthalmol 2010 Jan;13(1):37-42.
          pmc: PMC2930189pubmed: 20149174doi: 10.1111/j.1463-5224.2009.00755.xgoogle scholar: lookup
        26. Louboutin JP, Wang L, Wilson JM. Gene transfer into skeletal muscle using novel AAV serotypes.. J Gene Med 2005 Apr;7(4):442-51.
          pubmed: 15517544doi: 10.1002/jgm.686google scholar: lookup
        27. Gao G, Lu Y, Calcedo R, Grant RL, Bell P, Wang L, Figueredo J, Lock M, Wilson JM. Biology of AAV serotype vectors in liver-directed gene transfer to nonhuman primates.. Mol Ther 2006 Jan;13(1):77-87.
          pubmed: 16219492doi: 10.1016/j.ymthe.2005.08.017google scholar: lookup
        28. Netto MV, Mohan RR, Sinha S, Sharma A, Gupta PC, Wilson SE. Effect of prophylactic and therapeutic mitomycin C on corneal apoptosis, cellular proliferation, haze, and long-term keratocyte density in rabbits.. J Refract Surg 2006 Jun;22(6):562-74.
          pmc: PMC2756017pubmed: 16805119doi: 10.3928/1081-597x-20060601-08google scholar: lookup
        29. Mohan R. abstract. columbia, mo: Mason eye institute.
        30. Maltseva O, Folger P, Zekaria D, Petridou S, Masur SK. Fibroblast growth factor reversal of the corneal myofibroblast phenotype.. Invest Ophthalmol Vis Sci 2001 Oct;42(11):2490-5.
          pubmed: 11581188
        31. Morrison DF, Foss DL, Murtaugh MP. Interleukin-10 gene therapy-mediated amelioration of bacterial pneumonia.. Infect Immun 2000 Aug;68(8):4752-8.
          pmc: PMC98427pubmed: 10899882doi: 10.1128/iai.68.8.4752-4758.2000google scholar: lookup
        32. Sharma A, Ghosh A, Siddappa C. Ocular Surface: Gene Therapy. Encyclopedia of the Eye 2009 In Press.

        Citations

        This article has been cited 9 times.
        1. Mohan RR, Martin LM, Sinha NR. Novel insights into gene therapy in the cornea.. Exp Eye Res 2021 Jan;202:108361.
          doi: 10.1016/j.exer.2020.108361pubmed: 33212142google scholar: lookup
        2. 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(8):e0182473.
          doi: 10.1371/journal.pone.0182473pubmed: 28763501google scholar: lookup
        3. Marlo TL, Giuliano EA, Tripathi R, Sharma A, Mohan RR. Altering equine corneal fibroblast differentiation through Smad gene transfer.. Vet Ophthalmol 2018 Mar;21(2):132-139.
          doi: 10.1111/vop.12485pubmed: 28685927google scholar: lookup
        4. Ljubimov AV, Saghizadeh M. Progress in corneal wound healing.. Prog Retin Eye Res 2015 Nov;49:17-45.
          doi: 10.1016/j.preteyeres.2015.07.002pubmed: 26197361google scholar: lookup
        5. Mohan RR, Rodier JT, Sharma A. Corneal gene therapy: basic science and translational perspective.. Ocul Surf 2013 Jul;11(3):150-64.
          doi: 10.1016/j.jtos.2012.10.004pubmed: 23838017google scholar: lookup
        6. 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(10):e26432.
          doi: 10.1371/journal.pone.0026432pubmed: 22039486google scholar: lookup
        7. 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 Jul 1;52(7):4833-41.
          doi: 10.1167/iovs.11-7357pubmed: 21551414google scholar: lookup
        8. Mohan RR, Sharma A, Cebulko TC, Tandon A. Vector delivery technique affects gene transfer in the cornea in vivo.. Mol Vis 2010 Nov 27;16:2494-501.
          pubmed: 21139995
        9. Buss DG, Sharma A, Giuliano EA, Mohan RR. Efficacy and safety of mitomycin C as an agent to treat corneal scarring in horses using an in vitro model.. Vet Ophthalmol 2010 Jul;13(4):211-8.
          doi: 10.1111/j.1463-5224.2010.00782.xpubmed: 20618797google scholar: lookup
        Subscribe to our newsletter
        Join 99,344 horse owners like you who receive our email updates on horse health and nutrition.