Protein engineering, Central nervous system, Drug delivery, Blood-brain barrier, Transferrin receptor, Yeast surface display, Molecular trojan horse, Drug delivery system, Transferrin-Receptors, Yeast-Surfaces
The blood-brain barrier (BBB) protects the Central Nervous System (CNS) from potentially harmful compounds that circulate in the blood, presenting a major obstacle to delivering drugs intended to treat disorders of the CNS like Alzheimer’s Disease, Parkinson's’ Disease, or brain tumors. In order to transport specific molecules that are needed for CNS function into the brain, there are dedicated receptors on the BBB to facilitate active transport from the blood into the CNS. One strategy to circumvent the challenge of drug delivery to the CNS is the use of molecular Trojan horse proteins. Trojan horse proteins are non-native ligands for BBB receptors that have been engineered to bind an extracellular epitope on a particular BBB receptor. In binding to this receptor, these Trojan horse proteins exploit the receptor-mediated transport system to be taken up across the BBB by the same mechanism that the receptor’s native ligand is moved into the CNS from the blood. These molecular Trojan horse proteins can then be conjugated to therapeutic drugs to facilitate the delivery of the entire drug complex. The purpose of this project was to engineer molecular Trojan horse proteins against the human transferrin receptor (TfR), a BBB receptor whose native function is in iron regulation. The long-term application of these engineered proteins is to conjugate them to therapeutic proteins for delivery across the BBB. The 10th type III domain of the human fibronectin (Fn3) protein was used as the scaffold protein in the construction of the engineered proteins. A library of Fn3 proteins with modifications made at their three surface binding loops was put into yeast cell surface display (YSD) format. Fn3 variants that bind to TfR were identified using magneticactivated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Results from this project show that the initial Fn3 library was successfully enriched using MACS and FACS for Fn3 variants that bind to soluble transferrin receptor (sTfR). The population isolated by these sorting techniques was then subjected to random mutagenesis in order to reintroduce diversity amongst the Fn3 variants in YSD format. The diversified secondgeneration Fn3 library was also successfully enriched for sTfR-binding variants using MACS and FACS. The Fn3 variants isolated from the sort rounds were then characterized using sequence analysis and titration binding assays. Moving forward, the engineered Fn3 variants that bind to TfR will be solubly produced and purified to allow for further characterization, including their binding to mammalian cells that express the TfR, internalization into these cells, and transport across the BBB. Ultimately, these molecular Trojan horse proteins will be conjugated to a variety of therapeutic cargo to aid in the treatment of CNS disorders.
Smith, Natalie Durham Sorkin, "Protein engineering for receptor-mediated drug delivery across the blood-brain barrier" (2016). Theses, Dissertations, and Projects. 1644.