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.
Mining a yeast library for brain endothelial cell-binding antibodies. Wang XX, Cho YK, Shusta EV. Nature methods. 2007; 4(2):143-5. NIHMSID: NIHMS88842 PubMed [journal]PMID: 17206151 PMCID: PMC2637222
Development of GFP-based biosensors possessing the binding properties of antibodies. Pavoor TV, Cho YK, Shusta EV. Proceedings of the National Academy of Sciences of the United States of America. 2009; 106(29):11895-900. PubMed [journal]PMID: 19574456 PMCID: PMC2715507
Human Blood-Brain Barrier Endothelial Cells Derived from Pluripotent Stem Cells. Lippmann E.S., Azarin S.M., Kay J.E., Nessler R.A., Wilson H.K., Al-Ahmad A., Palecek S.P., Shusta E.V. Nature Biotechnology, 30, 783-791, 2012.
Modeling the blood-brain barrier using stem cell sources. Lippmann ES, Al-Ahmad A, Palecek SP, Shusta EV. Fluids and barriers of the CNS. 2013; 10(1):2. PubMed [journal]PMID: 23305164 PMCID: PMC3564868
Identifying blood-brain-barrier selective single-chain antibody fragments. Jones AR, Stutz CC, Zhou Y, Marks JD, Shusta EV. Biotechnology journal. 2014; 9(5):664-74. NIHMSID: NIHMS584912 PubMed [journal]PMID: 24644233 PMCID: PMC4073886
A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources. Lippmann ES, Al-Ahmad A, Azarin SM, Palecek SP, Shusta EV. Scientific reports. 2014; 4:4160. PubMed [journal]PMID: 24561821 PMCID: PMC3932448
Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling. Lian X, Bao X, Al-Ahmad A, Liu J, Wu Y, Dong W, Dunn KK, Shusta EV, Palecek SP. Stem cell reports. 2014; 3(5):804-16. PubMed [journal]PMID: 25418725 PMCID: PMC4235141
Directed evolution of brain-derived neurotrophic factor for improved folding and expression in Saccharomyces cerevisiae. Burns ML, Malott TM, Metcalf KJ, Hackel BJ, Chan JR, Shusta EV. Applied and environmental microbiology. 2014; 80(18):5732-42. PubMed [journal]PMID: 25015885 PMCID: PMC4178591
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 PubMed [journal]PMID: 25340933 PMCID: PMC5051266
An evolved Mxe GyrA intein for enhanced production of fusion proteins. Marshall CJ, Grosskopf VA, Moehling TJ, Tillotson BJ, Wiepz GJ, Abbott NL, Raines RT, Shusta EV. ACS chemical biology. 2015; 10(2):527-38. PubMed [journal]PMID: 25384269 PMCID: PMC4340354
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. PubMed [journal]PMID: 25994964 PMCID: PMC4455681
Pro-region engineering for improved yeast display and secretion of brain derived neurotrophic factor. Burns ML, Malott TM, Metcalf KJ, Puguh A, Chan JR, Shusta EV. Biotechnology journal. 2016; 11(3):425-36. PubMed [journal]PMID: 26580314
Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation. Tillotson BJ, Goulatis LI, Parenti I, Duxbury E, Shusta EV. PloS one. 2015; 10(12):e0145820. PubMed [journal]PMID: 26713870 PMCID: PMC4694649
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 PubMed [journal]PMID: 27421304 PMCID: PMC5014630
Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier. Vatine GD, Al-Ahmad A, Barriga BK, Svendsen S, Salim A, Garcia L, Garcia VJ, Ho R, Yucer N, Qian T, Lim RG, Wu J, Thompson LM, Spivia WR, Chen Z, Van Eyk J, Palecek SP, Refetoff S, Shusta EV, Svendsen CN. Cell stem cell. 2017; 20(6):831-843.e5. PubMed [journal]PMID: 28526555
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. PubMed [journal]PMID: 27846787 PMCID: PMC5175444
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. 2017; 140(6):874-888. NIHMSID: NIHMS837416 PubMed [journal]PMID: 27935037 PMCID: PMC5339046