Mouse brain-wide transgene expression by systemic injection of genetically engineered exosomes: CAP-Exosomes

The bottleneck in drug discovery for central nervous system diseases is the absence of effective systemic drug delivery technology for delivering therapeutic drugs into the brain. Although some Adeno Associated Virus (AAV) serotype can cross blood brain barrier and deliver virus genome packaged therapeutic DNA (gene) or RNA molecules to brain cells along with other organs, several hurdles have emerged in the AAV9 vector gene transfer technology in both preclinical studies and clinical trials. In order to overcome some of the hurdles, we have developed a workflow to generate a novel brain targeted drug delivery system (DDS) that involves generation of genetically engineering exosomes by first selecting various functional AAV capsid specific peptides (collectively called CAP) known to be involved in brain targeted high expression gene delivery, and then expressing the CAP in frame with lysosome-associated membrane glycoprotein (Lamp2b) followed by expressing CAP-Lamp2b fusion protein on the surface of mesenchymal stem cell derived exosomes, generating CAP-exosomes. Intravenous injection of green fluorescent protein (GFP) gene loaded CAP-exosomes in mice transfer GFP gene throughout the CNS as measured by monitoring brain sections for GFP expression with confocal microscopy. GFP gene transfer efficiency is at least 20-fold greater than that of control Lamp2b-exosomes. GFP gene transduction to mouse liver was low. CAP-exosome has advantage over AAV-vector including, 1) no restriction in gene size to be delivered, 2) expected reduced production of neutralizing antibody, and 3) can be used separately for drug repurposing and/or in combination with therapeutic genes. Graphical Abstract