Nanoconjugates for normalization of cultured human diabetic limbal epithelial cells and organ-cultured corneas by gene therapy

Abstract

Purpose : To validate new gene therapy approach that would allow to deliver antisense oligonucleotides (AON) targeting diabetes-associated genes, to human diabetic stem cell-enriched cultured limbal epithelial cells (LEC) and organ-cultured corneas, using novel nanoconjugates based on polymalic acid (PMLA) scaffold and carrying AONs to cathepsin F and to miR-409 that would activate c-met expression.

Methods : LEC cultures and corneal organ cultures were established from postmortem human donor eyes. PMLA-based nanoconjugate contained cell targeting antibody to transferrin receptor, and morpholino AONs to cathepsin F (CF) and to miR-409-3p (both AON at 5 µM) that targets c-met proto-oncogene. Control nanoconjugate had a scrambled AON. Healing of heptanol-induced corneal epithelial wounds was monitored. Apoptosis was assessed with AnnexinV staining. Adenoviral transductions were performed as previously published. Various markers were assessed by immunostaining.

Results : Previously, we showed that epithelial cells in corneal organ culture were easily transduced with adenoviral (AV) constructs carrying c-met or CF shRNA. However, AV transduction of cultured LEC even at low level of the multiplicity of infection (80 pfu/cell) caused significant cytotoxicity (cell rounding and apoptosis). On the contrary, nanoconjugate with AON miR+CF used in cultured LEC at a wide dose range (5-30 µM AON) showed no significant differences in the numbers of AnnexinV-positive apoptotic cells (<5%) from untreated LEC or LEC treated with control nanoconjugates.
As expected, nanoconjugate with AON miR+CF treatment caused an increase of its target c-met and a decrease of CF protein levels in diabetic LEC and in organ-cultured diabetic corneas. This treatment also upregulated stem cell markers ABCG2 and keratin 15 in diabetic LEC. Similarly, stem cell (ABCG2, keratin 17 and ΔNp63) and diabetic (integrinα₃β₁and nidogen-1) markers were upregulated, and wound healing was significantly accelerated after nanoconjugate treatment of organ-cultured diabetic corneas. Treatment also normalized the expression of activated signaling intermediates, EGFR-Akt and p38 in the organ-cultured diabetic corneas.

Conclusions : Non-toxic nanoconjugates provide a new alternative to viral-based gene therapy in normalizing diabetic limbal epithelial cells and organ-cultured corneas.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.