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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.
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Publication Date: 2010-09-16 PubMed ID: 20840107PubMed Central: PMC3711113DOI: 10.1111/j.1463-5224.2010.00813.xGoogle Scholar: Lookup
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  • 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

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        Citations

        This article has been cited 11 times.
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