The blood vessel network of the brain is comprised of specialized endothelial cells that separate the bloodstream from the brain interior. These brain endothelial cells are so impermeable that the brain vasculature is oftentimes referred to as the blood-brain barrier (BBB). As a result of its barrier properties, the BBB plays an extremely important role in central nervous system (CNS) homeostasis by protecting neurons from fluctuations in blood composition and from toxic bloodborne substances. Although the endothelium provides the barrier properties of the BBB, it is the local brain microenvironment that elicits the unique phenotype. Vascular smooth muscle cells line precapillary arterioles; pericytes share a basement membrane with capillary endothelial cells; astrocytes ensheath the microvessels; and nerve terminals contact the endothelium. Together with the endothelium, these perivascular cell types constitute the so-called neurovascular unit (NVU). As a result of BBB barrier properties, non-invasive delivery of small molecule pharmaceuticals and biopharmaceuticals (protein pharmaceuticals) to the brain is limited. Unless a molecule satisfies the dual criteria of having a small molecular size of less than 600 daltons and a high degree of lipid solubility, it will not appreciably cross the BBB. Because of these constraints, greater than 98% of small molecule pharmaceuticals do not cross the BBB and no biopharmaceuticals can cross this barrier. We are focused on overcoming this barrier through the development of non-invasive delivery methods that target drugs to the brain for the treatment of neurological diseases. Traditionally, the design of neuropharmaceuticals has been chemistry-driven and has relied on the manipulation of small molecule compounds to satisfy the size and lipid solubility requirements. However, molecular engineering techniques allow us to take a different approach and employ endogenous transport mechanisms present at the BBB as a means to shuttle drug cargo from the blood to the brain. These cellular transport systems can be targeted using the exquisite specificity of antibodies that are in turn linked to a drug payload that can include small molecule pharmaceuticals, biopharmaceuticals, or even DNA therapeutics. We are therefore interested in the discovery of novel transport systems and cognate antibody targeting molecules, and we design high throughput selections that serve this purpose. Along these lines, we are also working to optimize the process for producing large amounts of therapeutic antibodies and proteins to meet the eventual demands of clinical application. We are also interested in developing in vitro models of the BBB that accurately mimic the in vivo characteristics of the BBB. An in vitro BBB model would permit the combinatorial screening of drug candidates and drug-targeting strategies, a process that is not amenable to an in vivo system. When the endothelial cells that make up the BBB are cultured in vitro, however, changes in gene and protein expression occur thereby altering the permeability characteristics and integrity of the in vitro model. We have investigated these changes using genomics and proteomics techniques in an attempt to understand how gene and protein expression must be modulated to yield properties representative of the in vivo BBB. We are working to leverage this information for the development of novel in vitro models that possess more in vivo-like qualities. To this end, we have recently deployed pluripotent stem cell technology to model the human BBB in health and disease. In addition to being able to predict drug permeability at the BBB, we are using patient-derived induced pluripotent stem cell technology to study the NVU in brain disease and identify antibodies capable of brain drug delivery.
Eric Shusta, PhD
Chemical and Biological Engineering
3631 Engineering Hall
1415 Engineering Dr
Madison, WI 53706
We are interested in the discovery of novel transport systems and cognate antibody targeting molecules, and we design high throughput selections that serve this purpose. Along these lines, we are also working to optimize the process for producing large amounts of therapeutic antibodies and proteins to meet the eventual demands of clinical application. We are also interested in developing in vitro models of the BBB that accurately mimic the in vivo characteristics of the BBB.
Cells and cell lysates: a direct approach for engineering antibodies against membrane proteins using yeast surface display. Tillotson BJ, Cho YK, Shusta EV. Methods (San Diego, Calif.). 2013; 60(1):27-37. NIHMSID: NIHMS369522 PMCID: PMC3405166
Creation and Evaluation of a Single-chain Antibody Tetramer that Targets Brain Endothelial Cells. Zhang X, Wang XX, Shusta EV. AIChE journal. American Institute of Chemical Engineers. 2014; 60(4):1245-1252. NIHMSID: NIHMS553501 PMCID: PMC3958949
Targeting receptor-mediated transport for delivery of biologics across the blood-brain barrier. Lajoie JM, Shusta EV. Annual review of pharmacology and toxicology. 2015; 55:613-31. NIHMSID: NIHMS817435 PMCID:PMC5051266
Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells. Wilson HK, Canfield SG, Hjortness MK, Palecek SP, Shusta EV. Fluids and barriers of the CNS. 2015; 12:13. PMCID:PMC4455681
Advances in microfluidic platforms for analyzing and regulating human pluripotent stem cells. Qian T, Shusta EV, Palecek SP. Current opinion in genetics & development. 2015; 34:54-60. NIHMSID: NIHMS713546 PMCID:PMC4552035
In vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use. Helms HC, Abbott NJ, Burek M, Cecchelli R, Couraud PO, Deli MA, Förster C, Galla HJ, Romero IA, Shusta EV, Stebbins MJ, Vandenhaute E, Weksler B, Brodin B. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2016; 36(5):862-90. PMCID:PMC4853841
Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells. Stebbins MJ, Wilson HK, Canfield SG, Qian T, Palecek SP, Shusta EV. Methods (San Diego, Calif.). 2016; 101:93-102. NIHMSID: NIHMS738012 PubMed [journal] PMCID:PMC4848177
Analysis of Cancer-Targeting Alkylphosphocholine Analogue Permeability Characteristics Using a Human Induced Pluripotent Stem Cell Blood-Brain Barrier Model. Clark PA, Al-Ahmad AJ, Qian T, Zhang RR, Wilson HK, Weichert JP, Palecek SP, Kuo JS, Shusta EV. Molecular pharmaceutics. 2016; 13(9):3341-9. NIHMSID: NIHMS804541
Cryopreservation of Brain Endothelial Cells Derived from Human Induced Pluripotent Stem Cells Is Enhanced by Rho-Associated Coiled Coil-Containing Kinase Inhibition. Wilson HK, Faubion MG, Hjortness MK, Palecek SP, Shusta EV. Tissue engineering. Part C, Methods. 2016; 22(12):1085-1094.
An Isogenic Blood-Brain Barrier Model Comprising Brain Endothelial Cells, Astrocytes and Neurons Derived from Human Induced Pluripotent Stem Cells. Canfield SG, Stebbins MJ, Morales BS, Asai SW, Vatine GD, Svendsen CN, Palecek SP, Shusta EV. Journal of neurochemistry. 2016;
Protein engineering approaches for regulating blood-brain barrier transcytosis. Goulatis LI, Shusta EV. Current opinion in structural biology. 2016; 45:109-115.