Construction of a Lentiviral System Harboring Kringle 1-5 and Endostatin::Kringle-5 and Expression in Rabbit Conjunctiva in a Rabbit Corneal Neovascularization Assay

Abstract

Abstract: : Purpose: To develop and test lentiviral vectors capable of promoting the expression of potential angiogenesis inhibitors. These vectors harbored genes which included either 1) the five kringle domains of human plasminogen (K1-5), or 2) a fusion gene containing Endostatin-18 (a 20kD protein product derived from the carboxy end of collagen 18) fused with Kringle-5 (the fifth kringle domain within human Angiostatin). Methods:A cDNA coding for the 452 amino acid protein K1-5 was cloned into the lentiviral vector pHR'-IRES-eGFP under the control of the EF-1α/HTLV promoter. A cDNA coding for the 313 amino acid of the EK-5 fusion protein was cloned into a pHR'-IRES-eGFP vector under the CMV promoter. EK-5 and K1-5 replication-defective lentivirus was prepared by three plasmid co-transfection into 293T cells. Transcription of this gene produces a bicistronic message, capable of the independent translation of EK-5/ K1-5 and eGFP proteins. Expression of EK-5 and K1-5 in human microvascular endothelial cells transduced with this virus was analyzed by RT-PCR analysis. To assay the angiogenic potential of this reagent, subconjunctival injections of either the pHR'-EF1α /HTLV-K1-5-IRES-eGFP or pHR'-CMV-EK-5-IRES-eFGP virus or pHR'-IRES-eGFP (a gene-deficient control) virus was given in the inferior conjunctiva of New Zealand White rabbits by a single blinded surgeon. Corneas were challenged with intrastromal silk sutures 14 days after transduction. The neovascular response was measured by mm and clock hours of neovascularization onto the cornea on days 3, 6, 9 and 18 days after insult by a single blinded observer. The contralateral eye served as a control with subconjuntival injection of PBS. Results: A fusion cDNA coding for Kringle1-5 and Endostatin::Kringle-5 was successfully cloned into a lentiviral plasmid pHR'IRES-eGFP vector. Despite the fact that the recovery of transduced tenon cells failed to demonstrate transgene expression by rtPCR, these reagents show the ability to inhibit neovascularization in rabbit corneal assay on days 6, 9, and 18 days after transduction with significance of p<.05. Conclusions: Kringle1-5 and Endostatin::Kringle-5 inhibit corneal neovascularization through lentiviral transduction via subconjunctival injection in a rabbit model.

Keywords: gene transfer/gene therapy • neovascularization • conjunctiva