TGFB1-induced extracellular expression of TGFBIp and inhibition of TGFBIp expression by RNA interference in a human corneal epithelial cell line.

Of the 35,000 to 40,000 corneal transplants performed in the United States annually, approximately 15% to 23% are performed for management of a corneal dystrophy.1–4 The genetic basis of two-thirds of the approximately 30 corneal dystrophies has been elucidated, with five of the most common dystrophies associated with dominant mutations in the transforming growth factor, β-induced gene (TGFBI; MIM 601692).5 Mutations in TGFBI result in the deposition of dysfunctional TGFBI protein (TGFBIp) in the corneal stroma in the form of discrete or confluent dystrophic deposits.6,7 If the dystrophic corneal deposits are superficially located, painful recurrent epithelial erosions may develop. Management of corneal stromal opacification or recurrent corneal erosions is typically achieved with laser phototherapeutic keratectomy (PTK), lamellar keratoplasty, or penetrating keratoplasty (PK). Although PTK is an effective technique to remove superficial dystrophic corneal deposits, it is not effective in a large percentage of patients with TGFBI dystrophies caused by the presence of visually significant deposits in the mid and posterior stroma. Additionally, PTK is associated with several potential complications such as induced corneal scarring, irregular astigmatism, and hyperopia. Penetrating and lamellar keratoplasty are also associated with a multitude of potential intraoperative and postoperative complications, including recurrence of the dystrophic deposits in the transplanted cornea, and thus are reserved for patients in whom more conservative therapies have failed. In patients with TGFBI corneal dystrophies, the dystrophic deposits typically recur after both PTK and PK.8–10 In four published series documenting the recurrence of the TGFBI dystrophies after PK, the percentage of patients who experienced recurrence of the dystrophic deposits in the transplanted cornea was approximately 43% for granular corneal dystrophy (GCD), 48% to 60% for lattice corneal dystrophy (LCD), and 88% to 100% for corneal dystrophy of Bowman layer type I (CBD I) and CBD II.10–13 The median time to recurrence is highly variable but was estimated to be approximately 2 years for CBD I and II and 8 years for LCD.10 The incidence and rate of recurrence of the TGFBI corneal dystrophies after PTK vary widely, likely secondary to the differences in the number of patients, lengths of follow-up, and definitions of recurrence in the various reports. If the data from each of the reports are combined, recurrent deposits developed in approximately 52% of patients with CDB I and II, 25% of patients with LCD, 38% of patients with GCD, and 100% of patients with combined granular lattice corneal dystrophy (CGLCD) after corneal transplantation.8,9,13–16 Mean time to recurrence was shortest in patients who were homozygous for the mutation associated with CGLCD (9.5 months) and in patients with CDB I and II (26 months). The need for surgical management of the dystrophic corneal deposits in most patients with TGFBI dystrophies has led to interest in nonsurgical means to prevent the development or the recurrence of the dystrophic corneal deposits, which may be associated with significant visual morbidity in affected patients. Immunohistochemical analysis of the corneal buttons removed from affected patients at the time of PK has shown that the deposits consist of mutated TGFBIp.17–20 Interestingly, dystrophic deposits are limited to the corneas of affected persons, as confirmed by clinical and autopsy studies.21 TGFBIp is constitutively produced by human corneal epithelial cells (HCECs),22 although the amount produced may be significantly increased in HCECs and in corneal stromal keratocytes in response to injury or surgery, as has been reported after LASIK procedures.23–28 We investigated the usefulness of RNA interference (RNAi) to inhibit or impede the formation of visually significant dystrophic corneal deposits in patients with TGFBI dystrophies and as a means to prevent the recurrence of visually significant dystrophic corneal deposits after PTK and corneal transplantation. Evidence to support the usefulness of this approach comes from previous in vitro and in vivo studies using RNAi to inhibit pathologic processes involving the cornea. This therapeutic strategy has been shown to significantly suppress the expression of vascular endothelial growth factor (VEGF) induced by hypoxia in HCECs and to inhibit injury-induced murine corneal neovascularization in vivo.29 RNAi has also been used in in vitro (cultured human corneal fibroblasts) and in vivo murine models of ocular inflammation to suppress the activity of TGF-β.30,31 In addition, RNAi has been shown to effectively decrease the expression of TGFBIp in an experimentally transformed cell line (HEK 293) transfected with plasmids expressing TGFBIp and TGFBIp-specific shRNA-generating plasmids.32 We report the efficacy of RNAi in decreasing TGFBIp expression in both TGFB1-induced and noninduced HCECs, which are the primary source of the dystrophic TGFBIp in persons with a TGFBI dystrophy.