182. Towards the Correction of the Genetic Defect in Corneal Keratinocytes from Patients with Macular Corneal Dystrophy Type II

Abstract

Top of pageAbstract Macular Corneal Dystrophy (MCD) is an autosomal recessive hereditary disease in which progressive punctuate opacities in the cornea lead to bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes (I and II) defined by the respective absence and presence of sulphated Keratan (KS) in the patient serum, although both types are clinically indistinguishable. The carbohydrate sulfotransferase 6 gene (CHST6) encoding corneal N-acetylglucosamine 6-O-sulfotransferase (C-GlcNAc6ST) has been identified as a cause of MCD I and II. C-GlcNAc6ST transfers sulphate residues to keratan, a carbohydrate moiety of the major proteoglycans in the corneal stroma responsible for maintaining corneal transparency. In MCD type II, deletions and/or replacements in the upstream region of CHST6 lead to loss of cornea-specific expression of CHST6. A new packaging cell line producing a retroviral vector expressing CHST6 was developed. We constructed a pL(CHST6-IRES-GFP)SN plasmid by cloning in a pLXSN backbone the CHST6 cDNA and a cassette containing the Internal Ribosome Entry Site (IRES) and the reporter gene GFP. Since no antibodies against C-GlcNAc6ST are available the IRES sequence assures that transduced cells express both GFP and the CHST6 gene. The Am12/L(CHST6-IRES-GFP)SN producer cell line was generated by transfection of the GP+E86 cell line followed by infection of the amphotropic packaging cell line GP+env Am12. GFP(+)-Am12 cells were sorted by FACS and cloned by limiting dilution. Clones with higher viral titres were analysed for lack of re-arrangements and number of integrated proviruses (4.3 kb in size) by Southern blotting. We will show that keratinocytes from corneal biopsies of patients with MCD II plated onto a feeder layer composed of 3T3-J2 and Am12/L(CHST6-IRES-GFP)SN can be infected. Transduced GFP(+) keratinocytes will be further analysed and classified as holo-, mero- or paraclones for their growth potential. To evaluate whether GFP(+) keratinocytes also express the CHST6 gene, the ability of the C-GlcNAc6ST enzyme to transfer sulphate residues to KS will be analysed by (i) keratan sulphate ELISA or (ii) HPLC after treatment of the polysaccharide with specific enzymes. The latter allows to determine both the amount of sulphated keratan (sensitivity down to 100 ng with bovine corneal KS) and the position to which the sulphation process occurs on the keratan backbone. Analysis of the sulphation process is fundamental in the case of MCD II in which sulphation is not absent (as in MCD I) but only reduced. To our knowledge this is the first report of attempting gene correction of corneal keratinocytes stem cells for correction of corneal hereditary diseases. Following the tremendous progress achieved in restoring damaged corneal surfaces by cultivation of autologous limbal stem cells (Pellegrini et al., Lancet 1997; 349: 990–993; Rama et al., Transplantation 2001; 72: 1478–1485), application of gene transfer protocols to autologous limbal keratinocytes cultured in vitro could represent a challenging alternative therapy for patients with MCD II.