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. 2010 Sep;13(5):301-6.
doi: 10.1111/j.1463-5224.2010.00813.x.

Gene delivery in the equine cornea: a novel therapeutic strategy

Dylan G Buss  1 , Elizabeth Giuliano, Ajay Sharma, Rajiv R Mohan
Affiliations

Gene delivery in the equine cornea: a novel therapeutic strategy

Dylan G Buss et al. Vet Ophthalmol. 2010 Sep.

Abstract

Objective: 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.

Material: Primary ECF cultures were harvested from healthy donor equine corneas. Cultures were maintained at 37°C in humidified atmosphere with 5% CO(2).

Procedure: 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.

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Figures

Figure 1
Figure 1
Light microscopy images of both ECFs alone and ECFs after application of AAV5 at two time points (24 hrs and 48 hrs post application). All images demonstrate the classic normal, spindle shape morphology of ECFs. ECFs alone at 24 hrs (A), ECFs + AAV5, 24 hrs after application of AAV5 (B), ECFs alone at 48 hrs (C), and ECFs + AAV5, 48 hrs after application of AAV5 (D). Calibration bar is 100 microns.
Figure 2
Figure 2
Percent viability of control ECFs and ECFs transduced with AAV5 over three time points: 8 hrs, 24 hrs and 48 hrs after AAV5 application.
Figure 3
Figure 3
Panels A–C demonstrate the successful transduction of ECF by AAV5 as evidenced by the expression of EGFP demonstrating the classic spindle shape cellular morphology. The ECF nuclei are stained by blue by DAPI. Panel D shows the quantification of the data using Image J software and expressed as percent transduction efficiency.
See this image and copyright information in PMC

References

    1. Mohan RR, Schultz GS, Hong JW, et al. Gene transfer into rabbit keratocytes using AAV and lipid-mediated plasmid DNA vectors with a lamellar flap for stromal access. Exp Eye Res. 2003;76:373–383. - PubMed
    1. Mohan RR, Sharma A, Netto MV, et al. Gene therapy in the cornea. Prog Retin Eye Res. 2005;24:537–559. - PubMed
    1. Verma IM, Weitzman MD. Gene therapy: twenty-first century medicine. Annu Rev Biochem. 2005;74:711–738. - PubMed
    1. Monahan PE, Samulski RJ. Adeno-associated virus vectors for gene therapy: more pros than cons? Mol Med Today. 2000;6:433–440. - PubMed
    1. Daya S, Berns KI. Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev. 2008;21:583–593. - PMC - PubMed

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