Keratoconus (KTCN), a non-inflammatory corneal disorder characterized by stromal thinning, represents a major cause of corneal transplantations. Genetic and environmental factors have a role in the etiology of this complex disease. Previously reported linkage analysis revealed that chromosomal region 13q32 is likely to contain causative gene(s) for familial KTCN. Consequently, we have chosen eight positional candidate genes in this region: MBNL1, IPO5, FARP1, RNF113B, STK24, DOCK9, ZIC5 and ZIC2, and sequenced all of them in 51 individuals from Ecuadorian KTCN families and 105 matching controls. The mutation screening identified one mutation and three sequence variants showing 100% segregation under a dominant model with KTCN phenotype in one large Ecuadorian family. These substitutions were found in three different genes: c.2262A4C (p.Gln754His) and c.720+43A4G in DOCK9; c.2377-132A4C in IPO5 and c.1053+29G4C in STK24. PolyPhen analyses predicted that c.2262A4C (Gln754His) is possibly damaging for the protein function and structure. Our results suggest that c.2262A4C (p.Gln754His) mutation in DOCK9 may contribute to the KTCN phenotype in the large KTCN-014 family.

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Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic.

Magnetofection is a promising tool for gene therapy of corneal endothelial cells with potential for targeted on-site delivery.

Corneal transplantation (keratoplasty) is the most common type of tissue replacement in the world. The increased rate of graft rejection after keratoplasty is a central problem for repeated transplantations and in inflamed host corneas. It has been shown that apoptosis of grafted epithelium has a role in corneal allograft rejection. This study focused on the T-cell response triggered in BALB/c mice after allogeneic corneal transplantation with and without anti-apoptotic p35-transduced epithelium. To restrict p35 expression to the epithelial cells, modified allogeneic composite grafts were created. As a result, it was found that the proportion of alloreactive CD4 T cells in postoperatively removed cervical lymph nodes was reduced in the p35-transduced group compared to the allogeneic control group. Diminished priming of the CD4 T cells was supported by significantly decreased proliferation and lower interferon gamma secretion when compared to allogeneic engraftments. The reduced priming of CD4 lymphocytes is the first confirmation of the functionality of p35 in the epithelium of corneal grafts to alter the development of the recipient's immune response. Thus, modification of allosensibilization seems to be a promising tool for reducing graft-mediated immune response following corneal transplantation.

A tissue-like scaffold mimicking the human stroma could be developed. The results indicate that PGS/PCL scaffolds could be considered as ideal candidates for corneal tissue engineering as they are biocompatible in contact to corneal endothelial cells and blood cells.