The blood brain barrier (BBB) and the blood cerebrospinal fluid barrier (BCSFB) preclude, under healthy conditions, the free exchange of biologicals between blood and brain. These barriers are of vital importance but severely hamper the delivery of drugs targeting central nervous system (CNS) related disorders or brain tumors. Promising strategies to cross the BBB/BCSFB, include drug delivery by means of targeting to the BBB/BCSFB endothelium, followed by effective transport of the delivery system across the BBB/BCSFB and subsequent release at the brain. In this process, known as receptor mediated transcytosis (RMT), specific receptors responsible for blood-to-brain delivery of nutrients are targeted using affinity binders. Proof of principle for RMT has been generated using antibodies targeting a BBB transporter, but further development has halted due to targetspecific safety concerns. We propose two pioneering and unbiased methods to identify new, safer and more efficient RMT targets for both BBB and BCSFB. Our innovative experimental design will result in the discovery of novel, RMT-mediating single-chain antibodies (VHHs) that we will further develop into efficient BBB/BCSFB shuttling moieties. In vivo proof-of-concept in mice will be obtained by coupling a set of promising candidates to a therapeutic antibody (cancer domain) and testing them in relevant mouse models. Early inclusion of mechanism-of-action studies and toxicity readouts will feed into the projected safety profile of our candidates. We will benchmark the best candidates in rhesus monkeys against a published, industry-developed compound to enable proper valuation of the assets generated in our project. Discovery of BBB/BCSFB-shuttling receptors with improved capacity and with a better safety profile and VHHS shuttling through them would be a major breakthrough in the treatment of CNS related diseases such as Alzheimer’ Disease, brain cancer and Multiple Sclerosis.