University of Virginia,
Keywords: exosome, gene delivery, antisense therapy, tumor targeting
Summary:Nucleic acid delivery by functionalized exosomes Jack Vietmeyer1, 2, Bowen Dong1, 3, Tao Huang1, 4, Jiang He1, 5 1 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA 22903 2 Department of Mechanic engineering, University of Virginia, Charlottesville, VA 22908 3 Albemarle High School, Charlottesville, VA 22901 4 Department of Pharmacology, University of Virginia, Charlottesville, VA 22908 5 UVa comprehensive Cancer Center, University of Virginia, Charlottesville, VA 22908 Introduction: Exosomes are extracellular nanometer-sized vesicles released through various cells. These vesicles serve as nanocarriers, possessing great potential to overcome some obstacles encountered in gene and drug delivery due to their natural affinity to recipient cells and the inherent capability to shuttle the genes, lipids, proteins, and RNAs between cells. This study is to develop a novel tumor targeting approach for exosome based delivery of antisense oligonucleotides. Methods: Exosomes were prepared from Fat free milk by sequential filtration with 450nm, 220nm, and centrifugation and precipitation. The quality of exosomes was confirmed and validated by HPLC and microscopy and ELISA for protein biomarkers. The exosomes were further functionalized with antisense oligonucleotides, asMORF to form exo-asMORF, and Cy5 for fluorescence imaging. RGD peptide was conjugated with sense oligonucleotides, MORF as tumor targeting agent. In vitro tumor targeting were assessed by incubating tumor cells with RGD conjugated with sense strand oligonucleotides (RGD-MORF) followed by exo-asMORF labeled with Cy5. In vivo studies of tumor targeting were performed in nude mice with PC3-luc tumors in prostate with co-administration of RGD-MORF and exo-asMORF along with control groups. Results: The exo-asMORF showed specific binding to tumor cells through binding to RGD conjugated sense MORF. In vivo tumor targeting was established by fluorescence imaging with the study group receiving both RGD-MORF and exo-asMORF exhibits significant stronger signals compared to other two control groups. Conclusion: Exosomes carrying oligonucleotides were targeted to tumor in vitro and in vivo. These results suggest new strategy for nucleic acid delivery and warrant further study of the therapeutic effect in vivo.