Optimization of nanoconjugate delivery into human diabetic limbal epithelial cells and organ-cultured diabetic corneas for gene therapy

Abstract

Purpose : The purposes were to 1) develop nanoconjugates for effective delivery of antisense oligonucleotides (AON) to limbal epithelial cells (LEC) for gene therapy of diabetic keratopathy, and 2) evaluate the effect of nanoconjugate with AON to cathepsin F (increased in diabetes), and to miR-409 targeting c-met proto-oncogene (decreased in diabetes), in organ-cultured human diabetic corneas.

Methods : Stem cell-enriched LEC cultures and corneal organ cultures were obtained from postmortem human donor eyes. Rhodamine-labeled nanoconjugates based on polymalic acid (PMLA) scaffold were synthesized (1) and contained different peptides or a targeting antibody to transferrin receptor (TfR), and morpholino AON to cathepsin F and to miR-409-3p (both AON at 5 mM) that indirectly activates c-met expression. Control nanoconjugate had a scrambled sequence AON (Gene Tools). Uptake of labeled nanoconjugates was evaluated under inverted fluorescent microscope over time. Healing of heptanol-induced corneal epithelial wounds was monitored microscopically.

Results : We tested several nano constructs based on PMLA and PEG conjugates with different moieties for facilitated and targeted uptake by cultured LEC. The moieties included anti-TfR antibody and various peptides, such as penetratin, Angiopep2, iron-mimic peptide, MiniAp-4 and starPEG-(Arg)₉. Peptides and nanoconjugates had different ability to penetrate into cultured LEC and accumulate in the endosomal compartment. This ability correlated with the expression of the corresponding receptor on LEC, which was minimal for Angiopep2 due to low level of its receptor LRP1 in LEC. High TfR expression in LEC enabled the PMLA-conjugate with anti-TfR antibody to rapidly (within 2 h) enter the cells and get internalized. High uptake by LEC was observed with free but not PMLA-conjugated penetratin and starPEG-(Arg)₉. In organ-cultured diabetic human corneas, accelerated wound healing and putative stem cell marker expression was observed after Lipofectamine- but not sildenafil-facilitated transfection with PMLA nanoconjugate carrying AONs to miR-409 and cathepsin F.

Conclusions : Nanoconjugates with cell-targeting peptides may be used as a new tool for gene therapy in normalizing diabetic limbal epithelial cells and diabetic corneal wound healing.
1. Proc Natl Acad Sci USA, 2010;107:18143-18148.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.