Engineered Exosome Producer Lentivirus

The exosome producer third-generation Lentivirus plasmid was custom built by VectorBuilder. Inc., which contained a hPGK1 promoter driving expression of CD63-L7Ae and Connexin43 S368A and EF1α promoter driving ZFP362-PAM- C/D_box expression. The inclusion of protease cleavage site P2A between CD63-L7Ae and Connexin43 S368A ensured protein separation. Mutations in the bovine growth hormone polyadenylation (bgh-PolyA) transcription termination signal after CD63-L7Ae and Connexin43 S368A enable read-through and expression of complete lentiviral genomic RNA expressed from the RSV promoter. The faulty poly (A) signal allowed for CD63-L7Ae and Connexin43 S368A expression. To obtain pLV-EXOtic-ZFP, DNA fragment between Afe1 and Acc65I i.e., PAMt was removed, overhangs filled in using Klenow Fragment (NEB) and re-ligated. To build the pLV-EXOtic-nLuc vector, the pLV-EXOtic-ZPAMt vector was digested with NsiI and BstBI (NEB) to remove the ZPAMt and replaced with the nLuc gene using NEBuilder HiFi DNA Assembly Master Mix (NEB) according to the manufacturer’s instructions. The calcium phosphate method (Takara) was used to transfected HEK 293 T cells with a mixture of genome transfer plasmid and packaging plasmid: pRRE, pCMV.Rev, pMD2.G^{61 (link)}, at a ratio of 4:2:1:1^{62 (link)}. The lentivirus vector was used at an MOI of 5 to transduce Mesenchymal Stem cells via spinoculation.