The goal of this thesis project is to isolate and characterize exosomes that express anti-human CD7 (α-hCD7) single chain variable fragment (scFv), which will be further utilized for T celltargeted gene editing. To reach this goal, exosomes from HEK 293 cells and Jurkat cells were isolated and characterized with different methodologies. To obtain α-hCD7 scFv-expressing exosomes, the HEK 293 cells were subjected to transfection of custom-plasmid pDisplay-αhCD7 scFv, and the expression of α-hCD7 scFv on the HEK 293-derived exosomes were then confirmed by immunogold staining, western blotting, and flow cytometry. To transfect 293 cells, calcium phosphate transfection was utilized, and the exosomes secreted by these cells were isolated via sequential ultracentrifugation steps. The exosomes isolated from the transfected cells were further characterized by a series of methodologies, including western blotting, flow cytometry, and immunogold labeling, to determine the classic exosome-specific markers along with the presence of α-hCD7 scFv on the surface of the exosomes. The results suggest the presence of α-hCD7 scFv on the surface of the transfected 293 cell as well as exosomes when compared to the untransfected HEK 293 cells and their exosomes. Furthermore, all exosomes isolated from HEK 293 and Jurkat cells expressed the typical exosome markers, such as CD63 and CD81. We further explored whether exosomes could hybrid with a liposome cargo to carry the CRISPR-Cas9 plasmid for gene editing. To achieve this, the exosomes isolated from 293 cells were subjected to hybrid formation with liposomes by simply mixing in cold temperatures. However, no hybrid formation was observed by transmission electronic microscopy (TEM). Taken together, in this thesis project, we have developed the methodologies to isolate and characterize the α-hCD7 scFv-expressing exosomes, further enabling the delivery of CRISPRCas9 using exosomes for efficient human T cell-targeted gene editing.
Date of publication
Guohua Yi, Rama Vankayalapati. Nagarjun Venkata, Mitsuo Ikebe, Jiyong Lee
Masters of Science in Biotechnology
Patel, Sahil, "ISOLATION AND CHARACTERIZATION OF α-HCD7 SCFV EXPRESSING EXOSOMES AND SYNTHESIS OF HYBRID EXOSOME-LIPOSOME FOR ANALYSES" (2022). Biotechnology Theses. Paper 1.