June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Evaluation of integrase-deficient vectors for lentiviral gene-transfer in corneal endothelium
Author Affiliations & Notes
  • Daniel Thieme
    Augenklinik, Universitatsklinikum Erlangen, Erlangen, Germany
  • Scott Ellis
    Oxford BioMedica, Oxford, United Kingdom
  • Thomas Armin Fuchsluger
    Augenklinik, Universitatsklinikum Erlangen, Erlangen, Germany
  • Friedrich E Kruse
    Augenklinik, Universitatsklinikum Erlangen, Erlangen, Germany
  • Footnotes
    Commercial Relationships   Daniel Thieme, None; Scott Ellis, Oxford BioMedica (E); Thomas Fuchsluger, None; Friedrich Kruse, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4085. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Daniel Thieme, Scott Ellis, Thomas Armin Fuchsluger, Friedrich E Kruse; Evaluation of integrase-deficient vectors for lentiviral gene-transfer in corneal endothelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4085.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose : Gene therapy approaches are explored to prevent EC apoptosis extending storage time of donor corneas and achieve post-operative grafts with higher EC quality. Retroviruses lead to an integration of the DNA of choice into the nucleus, a suboptimal feature in EC modification. We investigated the efficacy of integrase-deficient vector types as potential alternatives for EC transduction.

Methods : GFP-transducing vectors of EIAV and of HIV origin and their integrase-defective variants were used to transfer genes into human corneal endothelium. Corneal grafts were gained from donors of different age and gender (age 45 ± 24) and stored hypothermic (SightLife, Seattle). Each quarter of a cut cornea was transduced with the respective vector variants for 24h at 37°C. Fluorescence microscopy was done at 37°C with a Zeiss Spinning Disc confocal microscope endothelium-down. Amount of green fluorescence was quantified via ImageJ. Additionally, life imaging via intravital microscopy with non-linear optics and ary scan technology was used to establish fluorescence imaging in full thickness human corneas.

Results : Our studies showed that human corneas can be transduced with all used variations of lentiviral vectors namely EIAV (28 ±9%), EIAV integrase-defective (38 ±10%), HIV (17 ±7.0%) and HIV integrase deficient (24 ±7.5%). We noticed that integrase-deficient vectors transduced EC more effective than standard integrating vectors (p<0.05). Furthermore, we transduced a complete uncut cornea from the endothelial side and established life cell imaging throughout all layers of the cornea. We could visualize healthy and apoptotic as well as transduced corneal endothelial cells. The transduction efficiency for the corneal endothelium was 75% (± 9%) representing the higher vector to cell proportion in the "cornea-in-a-cup" transduction and we can state that only the endothelium was transduced showing the high specificity of our method.

Conclusions : With integrase-deficient vectors performing better in the corneal endothelium, a crucial step towards patient treatment is achieved considering regulatory questions. To our knowledge, this study shows for the first time in the corneal endothelium that integrase-deficient vectors work as effective as or better than their integrating counterparts. Given confirmation of these data in further experiments in human corneal tissue, we will transfer this concept into an eye setting.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×