Establishment of a HEK293T stable cell line capable of secreting GRP78-specific scFv-targeted nanovesicles

Exosomes are nanovesicular vehicles capable of transporting different cargoes. Based on their characteristics, exosomes are proposed as a class of vehicles for targeted delivery of therapeutics. We aimed to establish a HEK293T stable cell line capable of secreting GRP78-specific scFv-targeted exosomes.

The pLEX-LAMP2b-GRP78 construct was developed by enzymatic replacement of DARPin in pLEX-LAMP2b-DARPin with a GRP78-specific scFv. pLEX-LAMP2b-GRP78 (or TurboGFP as control), psPAX2, and pMD2.G plasmids were co-transfected into HEK293T cells, and produced lentiviruses were harvested. Different multiplicities of infection (MOI; 10, 20, 30, 60, 120, and 240) were used for the transduction of HEK293T to select the most appropriate one as assessed by flow cytometry. Transduced HEK293T cells were subject to puromycin selection and the presence of the scFv was assessed in the established cell line at the DNA, transcript, and protein levels by PCR, RT-PCR, and Western blotting, respectively.

pLEX-LAMP2b-GRP78 was successfully developed. Co-transfection resulted in the expression of GFP by HEK293T in the control group 48 hours following transfection. The MOI of 60 was selected as 10% of cells were GFP+ 72 hours following transduction. Following puromycin selection, the presence of the integrated scFv DNA and transcript was confirmed. Moreover, Western blotting results confirmed the presence of the His-tagged scFv in the established cell line.

HEK293T cells can be engineered for the production of targeted exosomes which could be applied for therapeutic purposes. Moreover, scFvs are potent targeting domains that could be leveraged for the development of targeted exosomes.