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Bhanu Dasari, Deepa Talreja, Siobhan Cashman, Rajendra Kumar-Singh; Aptamer Mediated Protein Delivery to Retinal Cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4551.
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© ARVO (1962-2015); The Authors (2016-present)
Therapeutic proteins have received prominent consideration in the treatment of various diseases. Exploitation of clinical potential of many therapeutic proteins for diseases of the eye is limited because of the challenges in overcoming ocular barriers including plasma membranes of cells. Because the cell membrane is mostly impermeable to proteins, protein therapeutics are limited to molecules that function extracellularly, e.g., antibodies. An efficient delivery system for transporting proteins to its intracellular target will provide expanded therapeutic modalities for protein-drugs. A multi-functional protein nucleolin acts as a shuttle between the plasma membrane and cytoplasm. Rapidly dividing cells express nucleolin on the membrane. Interestingly, nucleolin is also present on retinal cells. Aptamer AS1411 targets nucleolin and can be taken up by cells. Biotinylated AS1411 was exploited to deliver streptavidin to cells. We have also observed that AS1411/Streptavidin platform can be used to deliver therapeutic proteins. In this study, we wished to simplify the protein delivery system. We generated AS1411-Protein conjugates. We tested AS1411-GFP conjugate for uptake of GFP in the retina and AS1411-XIAP for caspase activity in the retina.
Protein-Oligo conjugation was based on two complementary heterobifunctional linkers, S-HyNic and S-4FB. Protein was modified with S-HyNic to form HyNic-Protein. Separately, amineAS1411 was modified with S-4FB to form 4FB-AS1411. The protein-AS1411 conjugate was made by mixing HyNic-Protein and 4FB-AS1411 in the presence of aniline. Intravitreal and subretinal injections of conjugates were carried out in BALB/c mice. Frozen sections were examined by fluorescence microscopy for GFP uptake in the retina. NMDA model of apoptosis was used to test XIAP delivery.
The conjugation of protein and AS1411 was confirmed by gel electrophoresis. Bis-aryl hydrazine bond formation between protein and oligo was confirmed by spectrophotometer readings at 354 nm. GFP uptake was observed throughout the retina in both intravitreal and subretinal injections of AS1411-GFP when compared with GFP injections. XIAP-AS1411 conjugate reduced Caspase 3/7 activity in NMDA treated eyes indicating XIAP delivery to cells.
The direct conjugation of AS1411 with proteins is simpler, and this method can be exploited to deliver therapeutic proteins such as antiapoptotic proteins or peptides to retinal cells.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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