UCARE: Undergraduate Creative Activities & Research Experiences

 

Date of this Version

Spring 4-7-2016

Document Type

Poster

Citation

American Society for Biochemistry and Molecular Biology annual meeting 2016; University of Nebraska-Lincoln Research Fair poster, Spring 2016.

Comments

Copyright (c) 2016 Colton M. Miller, Aaron J. Donner, Emma K. Blank, Andrew W. Egger, Brianna M. Keller, Punit P. Seth, and Edward N. Harris

Abstract

Introduction: Antisense oligonucleotides (ASOs) are short chemically modified oligonucleotides (5-7.4 kDa) that can produce a pharmacological effect by binding to RNA and affecting intermediary metabolism. Over 35 phosphorothioate (PS) ASOs are at various stages of clinical development for use as therapeutic agents and pharmacological tools. Antisense therapy is a progressing area of research, as these small strands of nucleotide oligomers can be produced to silence genes that aggravate chronic disorders or infections. An important distinction for ASOs compared to DNA is the substitution of the phosphodiester (PO) backbone with the PS modification. This sulfur substitution allows for these polar polyanionic molecules to have high stability in biological fluids and selective binding to cell surfaces. Although there is a premium for clinical development of these short chain molecules, the current understanding of the pathways for their ability to traverse plasma membranes remains unresolved. Injected gymnotic ASOs have been shown to accumulate in the liver via the organ’s functional scavenging mechanism in highly endocytically active sinusoidal endothelial cells (SECs) and Kupffer cells (KC) compared to hepatocytes. Our work outlines how a non-DNA binding class of scavenger receptors known as Stabilins binds to, and internalizes these small PS ASOs. Methods: Primary cells from rat and mouse were isolated and cultured with 125I-ASOs. Stable cell lines expressing Stabilin-1 and two Stabilin-2 isoforms (315-HARE and 190-HARE), were used to analyze binding affinity, endocytosis and degradation of 125I-ASOs in the cell. Co-localization was also done to analyze trafficking of ASOs once internalized within the cell by use of fluorescent ASO and lysotracker. Results: It was determined that PS ASOs bind with high affinity to the Stabilin class receptors with the majority of internalization performed by clathrin-mediated endocytosis. Binding was determined to be dependent on proper folding of the receptor, along with relying on salt-bridge formation. Once inside the cell via the Stabilin receptors, co-localization analysis showed ASOs being trafficked for degradation in the lysosome. Increased internalization rates of an ASO targeting the non-coding RNA of malat-1, in the Stabilin-expressing cell lines reduced malat-1 expression more efficiently, indicating not all ASOs are trafficked to the lysosome after internalization. Conclusion: Our work shows that ASOs are internalized into the cells of the liver through clathrin-mediated endocytosis. The understanding of the pathway(s) for chemically modified ASO internalization and trafficking with cell surface receptors will aid in future clinical design of ASOs as therapeutic agents.

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