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Md Imam Uddin, Tyler C. Kilburn, John S Penn; In vivo molecular imaging of endoglin mRNA using anti-sense shRNA-lipid bioconjugates as predictive biomarkers of neovascularization. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6063. doi: https://doi.org/.
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To investigate the application of a novel strategy for transfection-free targeted delivery of short hairpin-RNA-lipid bioconjugates, incorporating an anti-sense sequence complimentary to endoglin (CD105) mRNA (AS-ENG-shRNA-lipid). After systemic injection, these lipid conjugates are chaperoned by albumin throughout the circulation and are efficiently delivered to ocular tissues. These lipid conjugates are rapidly internalized by cells, and they incorporate a dye that becomes fluorescently active upon hybridization to target endoglin mRNA allowing their use as optical imaging probes.
These imaging probes were designed and synthesized by conjugating diacyl lipid to short hairpin-RNA that incorporates an anti-sense sequence complimentary to endoglin mRNA and a fluorophore that is quenched by a dye quencher in a distance-dependent manner. AS-ENG-shRNA-lipid conjugates were investigated to visualize endogenous endoglin mRNA in proliferative retinal microvascular endothelial cells (RMEC) in culture and in pathologic neovascular retinal lesions in the oxygen-induced retinopathy (OIR) model. The fluorescence in situ hybridization (FISH) technique was used to characterize focal expression of endoglin mRNA in proliferative neovascular tufts.
These novel probes are sensitive to a single-mismatch in a target mRNA sequence and are highly sensitive to the target complementary oligonucleotide, resulting in strong fluorescence emission. The fluorescence signals from AS-ENG-shRNA-lipid conjugates allow for visualization of the target mRNA in cells and in pathologic neovascular tufts in the OIR retina. A non-sense probe (NS-shRNA-lipid conjugate) remains dark in these tissues, demonstrating the specificity of this method. These shRNA-lipid conjugates were not acutely toxic to the RMECs as assessed by live-dead assay. Used ex vivo, FISH confirmed the focal expression of endoglin mRNA in neovascular lesions in OIR.
Endoglin (CD105) mRNA exhibits increased expression in proliferating endothelial cells at the earliest stages of retinal NV development. In this study, a novel mRNA imaging method has been developed to visualize endoglin mRNA in vivo as a diagnostic tool to predict the onset of NV, track NV progression and evaluate NV response to therapy.Keywords: molecular imaging, endoglin mRNA, proliferative endothelial cells, neovascularization.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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