September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Novel Nanoconjugates for Gene Therapy Normalization of Cultured Human Diabetic Limbal Epithelial Cells
Author Affiliations & Notes
  • Andrei A Kramerov
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Pallavi Gangalum
    Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Hui Ding
    Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Julia Y Ljubimova
    Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Alexander V Ljubimov
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Andrei Kramerov, None; Pallavi Gangalum, None; Hui Ding, None; Julia Ljubimova, None; Alexander Ljubimov, None
  • Footnotes
    Support  R01 EY013431
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Andrei A Kramerov, Pallavi Gangalum, Hui Ding, Julia Y Ljubimova, Alexander V Ljubimov; Novel Nanoconjugates for Gene Therapy Normalization of Cultured Human Diabetic Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Our purpose was to normalize cultured human diabetic limbal epithelial cells (LEC) that have reduced expression of epithelial stem cell markers and slow wound healing using novel polymalic acid-based nanoconjugates inhibiting MMP-10 and cathepsin F (increased in diabetic corneas) and upregulating c-met (decreased in diabetic corneas).

Methods : LEC cultures enriched in stem cells were obtained from postmortem donor eyes. Nanoconjugates on natural-derived polymalic acid scaffold were synthesized as published (1) and contained a targeting antibody to LEC-expressed transferrin receptor (TfR), morpholino antisense oligos (AON) to cathepsin F and to miR-409-3p that targets c-met proto-oncogene, and trileucine endosome escape unit. A separate nanoconjugate contained an AON to MMP-10 and a tracking dye Alexa Fluor 488. Efficiency of target inhibition (proteinases) or upregulation (c-met) was tested with free AON and then with whole nanoconjugates (at 5 mM AON). Healing of scratch wounds was monitored microscopically.

Results : Diabetic LEC from several donors were TfR-positive on western blots justifying the use of respective antibody for cell targeting. LEC internalized nanoconjugates mainly through receptor-mediated endocytosis as revealed by competition with free TfR antibody. By western blot, they were able to reduce the expression of MMP-10 and cathepsin F, and increase the expression of c-met (through inhibition of miR-409). Treatment with nanoconjugates accelerated healing of scratch wounds in LEC cultures with no toxicity observed.

Conclusions : Non-toxic nanoconjugates appear to be a new viable alternative to viral-based gene therapy in normalizing the diabetic limbal epithelial cells and wound healing.
1. Proc Natl Acad Sci USA, 2010;107:18143-18148.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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