PURPOSE: Gene-targeting technology and tissue-specific gene promoters were used to produce widespread expression of an exogenous gene throughout the eye, including the retinal pigmented epithelium, after noninvasive intravenous administration of a nonviral plasmid formulation. METHODS: An expression plasmid encoding bacterial beta-galactosidase, under the influence of either the simian virus (SV)40 promoter or the glial fibrillary acidic protein (GFAP) gene promoter, was packaged in the interior of 85-nm pegylated immunoliposomes (PIL) targeted to transferrin receptor (TfR)-rich structures with the rat 8D3 monoclonal antibody (mAb) to the mouse TfR. Plasmid DNA was packaged in the 8D3-PIL and injected intravenously into adult female BALB/c mice at a dose of 5 micro g DNA per mouse. The eyes were removed 48 hours later, and frozen sections were prepared for beta-galactosidase histochemistry and rhodopsin, TfR, or GFAP immunocytochemistry. RESULTS: There was diffuse expression of the SV40/beta-galactosidase gene in the retinal pigmented epithelium. The cellular specificity of gene expression was influenced by the promoter used in the gene construct, evidenced by the fact that gene expression in the inner retina was induced with the GFAP promoter. The beta-galactosidase gene was also widely expressed in the TfR-rich epithelial structures of the eye, including the ciliary body, the iris, the sebaceous glands of the tarsal plate, and the epithelium of the cornea. When the anti-TfR mAb on the PIL was replaced with the rat IgG isotype control, there was no gene expression in the eye. CONCLUSIONS: Widespread expression of an exogenous gene throughout the retina pigmented epithelium or other structures of the eye is possible with a noninvasive intravenous administration of a nonviral plasmid that is reformulated with gene-targeting technology. The cellular specificity of gene expression in the eye can be regulated with the use of cell-specific gene promoters.
PURPOSE: Gene-targeting technology and tissue-specific gene promoters were used to produce widespread expression of an exogenous gene throughout the eye, including the retinal pigmented epithelium, after noninvasive intravenous administration of a nonviral plasmid formulation. METHODS: An expression plasmid encoding bacterial beta-galactosidase, under the influence of either the simian virus (SV)40 promoter or the glial fibrillary acidic protein (GFAP) gene promoter, was packaged in the interior of 85-nm pegylated immunoliposomes (PIL) targeted to transferrin receptor (TfR)-rich structures with the rat 8D3 monoclonal antibody (mAb) to the mouseTfR. Plasmid DNA was packaged in the 8D3-PIL and injected intravenously into adult female BALB/c mice at a dose of 5 micro g DNA per mouse. The eyes were removed 48 hours later, and frozen sections were prepared for beta-galactosidase histochemistry and rhodopsin, TfR, or GFAP immunocytochemistry. RESULTS: There was diffuse expression of the SV40/beta-galactosidase gene in the retinal pigmented epithelium. The cellular specificity of gene expression was influenced by the promoter used in the gene construct, evidenced by the fact that gene expression in the inner retina was induced with the GFAP promoter. The beta-galactosidase gene was also widely expressed in the TfR-rich epithelial structures of the eye, including the ciliary body, the iris, the sebaceous glands of the tarsal plate, and the epithelium of the cornea. When the anti-TfR mAb on the PIL was replaced with the rat IgG isotype control, there was no gene expression in the eye. CONCLUSIONS: Widespread expression of an exogenous gene throughout the retina pigmented epithelium or other structures of the eye is possible with a noninvasive intravenous administration of a nonviral plasmid that is reformulated with gene-targeting technology. The cellular specificity of gene expression in the eye can be regulated with the use of cell-specific gene promoters.
Authors: Dylan E Parsons; Soo Hyeon Lee; Young Joo Sun; Gabriel Velez; Alexander G Bassuk; Mark Smith; Vinit B Mahajan Journal: Biomolecules Date: 2021-02-24