The proteome of the blood-brain barrier in rat and mouse: highly specific identification of proteins on the luminal surface of brain microvessels by in vivo glycocapture.

BACKGROUND: The active transport of molecules into the brain from blood is regulated by receptors, transporters, and other cell surface proteins that are present on the luminal surface of endothelial cells at the blood-brain barrier (BBB). However, proteomic profiling of proteins present on the luminal endothelial cell surface of the BBB has proven challenging due to difficulty in labelling these proteins in a way that allows efficient purification of these relatively low abundance cell surface proteins. METHODS: Here we describe a novel perfusion-based labelling workflow: in vivo glycocapture. This workflow relies on the oxidation of glycans present on the luminal vessel surface via perfusion of a mild oxidizing agent, followed by subsequent isolation of glycoproteins by covalent linkage of their oxidized glycans to hydrazide beads. Mass spectrometry-based identification of the isolated proteins enables high-confidence identification of endothelial cell surface proteins in rats and mice. RESULTS: Using the developed workflow, 347 proteins were identified from the BBB in rat and 224 proteins in mouse, for a total of 395 proteins in both species combined. These proteins included many proteins with transporter activity (73 proteins), cell adhesion proteins (47 proteins), and transmembrane signal receptors (31 proteins). To identify proteins that are enriched in vessels relative to the entire brain, we established a vessel-enrichment score and showed that proteins with a high vessel-enrichment score are involved in vascular development functions, binding to integrins, and cell adhesion. Using publicly-available single-cell RNAseq data, we show that the proteins identified by in vivo glycocapture were more likely to be detected by scRNAseq in endothelial cells than in any other cell type. Furthermore, nearly 50% of the genes encoding cell-surface proteins that were detected by scRNAseq in endothelial cells were also identified by in vivo glycocapture. CONCLUSIONS: The proteins identified by in vivo glycocapture in this work represent the most complete and specific profiling of proteins on the luminal BBB surface to date. The identified proteins reflect possible targets for the development of antibodies to improve the crossing of therapeutic proteins into the brain and will contribute to our further understanding of BBB transport mechanisms.

Proteome Profiling of Brain Vessels in a Mouse Model of Cerebrovascular Pathology.

Cerebrovascular pathology that involves altered protein levels (or signaling) of the transforming growth factor beta (TGFß) family has been associated with various forms of age-related dementias, including Alzheimer disease (AD) and vascular cognitive impairment and dementia (VCID). Transgenic mice overexpressing TGFß1 in the brain (TGF mice) recapitulate VCID-associated cerebrovascular pathology and develop cognitive deficits in old age or when submitted to comorbid cardiovascular risk factors for dementia. We characterized the cerebrovascular proteome of TGF mice using mass spectrometry (MS)-based quantitative proteomics. Cerebral arteries were surgically removed from 6-month-old-TGF and wild-type mice, and proteins were extracted and analyzed by gel-free nanoLC-MS/MS. We identified 3602 proteins in brain vessels, with 20 demonstrating significantly altered levels in TGF mice. For total and/or differentially expressed proteins (p ≤ 0.01, ≥ 2-fold change), using multiple databases, we (a) performed protein characterization, (b) demonstrated the presence of their RNA transcripts in both mouse and human cerebrovascular cells, and (c) demonstrated that several of these proteins were present in human extracellular vesicles (EVs) circulating in blood. Finally, using human plasma, we demonstrated the presence of several of these proteins in plasma and plasma EVs. Dysregulated proteins point to perturbed brain vessel vasomotricity, remodeling, and inflammation. Given that blood-isolated EVs are novel, attractive, and a minimally invasive biomarker discovery platform for age-related dementias, several proteins identified in this study can potentially serve as VCID markers in humans.

Preclinical Development and Characterization of Novel Adeno-Associated Viral Vectors for the Treatment of Lipoprotein Lipase Deficiency.

Lipoprotein lipase deficiency (LPLD) results from mutations within the lipoprotein lipase (LPL) gene that lead to a complete lack of catalytically active LPL protein. Glybera was one of the first adeno-associated virus (AAV) gene replacement therapy to receive European Medicines Agency regulatory approval for the treatment of LPLD. However, Glybera is no longer marketed potentially due to a combination of economical, manufacturing, and vector-related issues. The aim of this study was to develop a more efficacious AAV gene therapy vector for LPLD. Following preclinical biodistribution, efficacy and non-Good Laboratory Practice toxicity studies with novel AAV1 and AAV8-based vectors in mice, we identified AAV8 pVR59. AAV8 pVR59 delivered a codon-optimized, human gain-of-function hLPLS447X transgene driven by a CAG promoter in an AAV8 capsid. AAV8 pVR59 was significantly more efficacious, at 10- to 100-fold lower doses, compared with an AAV1 vector based on Glybera, when delivered intramuscularly or intravenously, respectively, in mice with LPLD. Efficient gene transfer was observed within the injected skeletal muscle and liver following delivery of AAV8 pVR59, with long-term correction of LPLD phenotypes, including normalization of plasma triglycerides and lipid tolerance, for up to 6 months post-treatment. While intramuscular delivery of AAV8 pVR59 was well tolerated, intravenous administration augmented liver pathology. These results highlight the feasibility of developing a superior AAV vector for the treatment of LPLD and provide critical insight for initiating studies in larger animal models. The identification of an AAV gene therapy vector that is more efficacious at lower doses, when paired with recent advances in production and manufacturing technologies, will ultimately translate to increased safety and accessibility for patients.

Differential Expression of ABC Transporter Genes in Brain Vessels vs. Peripheral Tissues and Vessels from Human, Mouse and Rat.

BACKGROUND: ATP-binding cassette (ABC) transporters comprise a superfamily of genes encoding membrane proteins with nucleotide-binding domains (NBD). These transporters, including drug efflux across the blood-brain barrier (BBB), carry a variety of substrates through plasma membranes against substrate gradients, fueled by hydrolyzing ATP. The expression patterns/enrichment of ABC transporter genes in brain microvessels compared to peripheral vessels and tissues are largely uncharacterized. METHODS: In this study, the expression patterns of ABC transporter genes in brain microvessels, peripheral tissues (lung, liver and spleen) and lung vessels were investigated using RNA-seq and WesTM analyses in three species: human, mouse and rat. RESULTS: The study demonstrated that ABC drug efflux transporter genes (including ABCB1, ABCG2, ABCC4 and ABCC5) were highly expressed in isolated brain microvessels in all three species studied; the expression of ABCB1, ABCG2, ABCC1, ABCC4 and ABCC5 was generally higher in rodent brain microvessels compared to those of humans. In contrast, ABCC2 and ABCC3 expression was low in brain microvessels, but high in rodent liver and lung vessels. Overall, most ABC transporters (with the exception of drug efflux transporters) were enriched in peripheral tissues compared to brain microvessels in humans, while in rodent species, additional ABC transporters were found to be enriched in brain microvessels. CONCLUSIONS: This study furthers the understanding of species similarities and differences in the expression patterns of ABC transporter genes; this is important for translational studies in drug development. In particular, CNS drug delivery and toxicity may vary among species depending on their unique profiles of ABC transporter expression in brain microvessels and BBB.

Mouse embryonic stem cell-derived blood-brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis.

Blood brain barrier (BBB) models in vitro are an important tool to aid in the pre-clinical evaluation and selection of BBB-crossing therapeutics. Stem cell derived BBB models have recently demonstrated a substantial advantage over primary and immortalized brain endothelial cells (BECs) for BBB modeling. Coupled with recent discoveries highlighting significant species differences in the expression and function of key BBB transporters, the field is in need of robust, species-specific BBB models for improved translational predictability. We have developed a mouse BBB model, composed of mouse embryonic stem cell (mESC-D3)-derived brain endothelial-like cells (mBECs), employing a directed monolayer differentiation strategy. Although the mBECs showed a mixed endothelial-epithelial phenotype, they exhibited high transendothelial electrical resistance, inducible by retinoic acid treatment up to 400 Ω cm2. This tight cell barrier resulted in restricted sodium fluorescein permeability (1.7 × 10-5 cm/min), significantly lower than that of bEnd.3 cells (1.02 × 10-3 cm/min) and comparable to human induced pluripotent stem cell (iPSC)-derived BECs (2.0 × 10-5 cm/min). The mBECs expressed tight junction proteins, polarized and functional P-gp efflux transporter and receptor mediated transcytosis (RMT) receptors; collectively important criteria for studying barrier regulation and drug delivery applications in the CNS. In this study, we compared transport of a panel of antibodies binding species selective or cross-reactive epitopes on BBB RMT receptors in both the mBEC and human iPSC-derived BEC model, to demonstrate discrimination of species-specific BBB transport mechanisms.

Epitope mapping of a blood-brain barrier crossing antibody targeting the cysteine-rich region of IGF1R using hydrogen-exchange mass spectrometry enabled by electrochemical reduction.

Pathologies of the central nervous system impact a significant portion of our population, and the delivery of therapeutics for effective treatment is challenging. The insulin-like growth factor-1 receptor (IGF1R) has emerged as a target for receptor-mediated transcytosis, a process by which antibodies are shuttled across the blood-brain barrier (BBB). Here, we describe the biophysical characterization of VHH-IR4, a BBB-crossing single-domain antibody (sdAb). Binding was confirmed by isothermal titration calorimetry and an epitope was highlighted by surface plasmon resonance that does not overlap with the IGF-1 binding site or other known BBB-crossing sdAbs. The epitope was mapped with a combination of linear peptide scanning and hydrogen-deuterium exchange mass spectrometry (HDX-MS). IGF1R is large and heavily disulphide bonded, and comprehensive HDX analysis was achieved only through the use of online electrochemical reduction coupled with a multiprotease approach, which identified an epitope for VHH-IR4 within the cysteine-rich region (CRR) of IGF1R spanning residues W244-G265. This is the first report of an sdAb binding the CRR. We show that VHH-IR4 inhibits ligand induced auto-phosphorylation of IGF1R and that this effect is mediated by downstream conformational effects. Our results will guide the selection of antibodies with improved trafficking and optimized IGF1R binding characteristics.

Grabody B, an IGF1 receptor-based shuttle, mediates efficient delivery of biologics across the blood-brain barrier.

Effective delivery of therapeutics to the brain is challenging. Molecular shuttles use receptors expressed on brain endothelial cells to deliver therapeutics. Antibodies targeting transferrin receptor (TfR) have been widely developed as molecular shuttles. However, the TfR-based approach raises concerns about safety and developmental burden. Here, we report insulin-like growth factor 1 receptor (IGF1R) as an ideal target for the molecular shuttle. We also describe Grabody B, an antibody against IGF1R, as a molecular shuttle. Grabody B has broad cross-species reactivity and does not interfere with IGF1R-mediated signaling. We demonstrate that administration of Grabody B-fused anti-alpha-synuclein (α-Syn) antibody induces better improvement in neuropathology and behavior in a Parkinson's disease animal model than the therapeutic antibody alone due to its superior serum pharmacokinetics and enhanced brain exposure. The results indicate that IGF1R is an ideal shuttle target and Grabody B is a safe and efficient molecular shuttle.

Brain Delivery of IGF1R5, a Single-Domain Antibody Targeting Insulin-like Growth Factor-1 Receptor.

The ability of drugs and therapeutic antibodies to reach central nervous system (CNS) targets is greatly diminished by the blood-brain barrier (BBB). Receptor-mediated transcytosis (RMT), which is responsible for the transport of natural protein ligands across the BBB, was identified as a way to increase drug delivery to the brain. In this study, we characterized IGF1R5, which is a single-domain antibody (sdAb) that binds to insulin-like growth factor-1 receptor (IGF1R) at the BBB, as a ligand that triggers RMT and could deliver cargo molecules that otherwise do not cross the BBB. Surface plasmon resonance binding analyses demonstrated the species cross-reactivity of IGF1R5 toward IGF1R from multiple species. To overcome the short serum half-life of sdAbs, we fused IGF1R5 to the human (hFc) or mouse Fc domain (mFc). IGF1R5 in both N- and C-terminal mFc fusion showed enhanced transmigration across a rat BBB model (SV-ARBEC) in vitro. Increased levels of hFc-IGF1R5 in the cerebrospinal fluid and vessel-depleted brain parenchyma fractions further confirmed the ability of IGF1R5 to cross the BBB in vivo. We next tested whether this carrier was able to ferry a pharmacologically active payload across the BBB by measuring the hypothermic and analgesic properties of neurotensin and galanin, respectively. The fusion of IGF1R5-hFc to neurotensin induced a dose-dependent reduction in the core temperature. The reversal of hyperalgesia by galanin that was chemically linked to IGF1R5-mFc was demonstrated using the Hargreaves model of inflammatory pain. Taken together, our results provided a proof of concept that appropriate antibodies, such as IGF1R5 against IGF1R, are suitable as RMT carriers for the delivery of therapeutic cargos for CNS applications.

Application of blood brain barrier models in pre-clinical assessment of glioblastoma-targeting CAR-T based immunotherapies.

Human blood brain barrier (BBB) models derived from induced pluripotent stem cells (iPSCs) have become an important tool for the discovery and preclinical evaluation of central nervous system (CNS) targeting cell and gene-based therapies. Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary form of gene-modified cell-based immunotherapy with potential for targeting solid tumors, such as glioblastomas. Crossing the BBB is an important step in the systemic application of CAR-T therapy for the treatment of glioblastomas and other CNS malignancies. In addition, even CAR-T therapies targeting non-CNS antigens, such as the well-known CD19-CAR-T therapies, are known to trigger CNS side-effects including brain swelling due to BBB disruption. In this study, we used iPSC-derived brain endothelial-like cell (iBEC) transwell co-culture model to assess BBB extravasation of CAR-T based immunotherapies targeting U87MG human glioblastoma (GBM) cells overexpressing the tumor-specific mutated protein EGFRvIII (U87vIII). Two types of anti-EGFRvIII targeting CAR-T cells, with varying tonic signaling profiles (CAR-F263 and CAR-F269), and control Mock T cells were applied on the luminal side of BBB model in vitro. CAR-F263 and CAR-F269 T cells triggered a decrease in transendothelial electrical resistance (TEER) and an increase in BBB permeability. CAR-T cell extravasation and U87vIII cytotoxicity were assessed from the abluminal compartment using flow cytometry and Incucyte real-time viability imaging, respectively. A significant decrease in U87vIII cell viability was observed over 48 h, with the most robust cytotoxicity response observed for the constitutively activated CAR-F263. CAR-F269 T cells showed a similar cytotoxic profile but were approximately four fold less efficient at killing the U87vIII cells compared to CAR-F263, despite similar transmigration rates. Visualization of CAR-T cell extravasation across the BBB was further confirmed using BBTB-on-CHIP models. The described BBB assay was able to discriminate the cytotoxic efficacies of different EGFRvIII-CARs and provide a measure of potential alterations to BBB integrity. Collectively, we illustrate how BBB models in vitro can be a valuable tool in deciphering the mechanisms of CAR-T-induced BBB disruption, accompanying toxicity and effector function on post-barrier target cells.

Immunoassay for Quantitative Detection of Antibody Transcytosis Across the Blood-Brain Barrier In Vitro.

Automated high-throughput immunoassays are emerging as reliable analytic techniques for the quantitative detection of proteins from a variety of sample types. Herein, we describe a method using the Protein Simple Wes capillary-based automated immunoassays platform for the quantification of His- and HA-tagged antibody transcytosis across an in vitro transwell blood-brain barrier (BBB) model. Compared to conventional ELISA, fluorescence, and Mass Spec-based detection approaches, Wes provides comparable datasets with additional information regarding size, aggregation, and potential degradation of samples before and after BBB transcytosis. In this chapter, we have benchmarked our Wes technique against ELISA and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), using known BBB crossing (FC5) and non-crossing (A20.1) single domain antibodies.

Targeting insulin-like growth factor-1 receptor (IGF1R) for brain delivery of biologics.

The blood-brain barrier (BBB) prevents the majority of drugs from crossing into the brain and reaching neurons. To overcome this challenge, safe and non-invasive technologies targeting receptor-mediated pathways have been developed. In this study, three single-domain antibodies (sdAbs; IGF1R3, IGF1R4, and IGF1R5) targeting the extracellular domain of the human insulin-like growth factor-1 receptor (IGF1R), generated by llama immunization, showed enhanced transmigration across the rat BBB model (SV-ARBEC) in vitro. The rate of brain uptake of these sdAbs fused to mouse Fc (sdAb-mFc) in vivo was estimated using the fluorescent in situ brain perfusion (ISBP) technique followed by optical brain imaging and distribution volume evaluation. Compared to the brains perfused with the negative control A20.1-mFc, the brains perfused with anti-IGF1R sdAbs showed a significant increase of the total fluorescence intensity (~2-fold, p < .01) and the distribution volume (~4-fold, p < .01). The concentration curve for IGF1R4-mFc demonstrated a linear accumulation plateauing at approximately 400 µg (~1 µM), suggesting a saturable mechanism of transport. Capillary depletion and mass spectrometry analyses of brain parenchyma post-ISBP confirmed the IGF1R4-mFc brain uptake with ~25% of the total amount being accumulated in the parenchymal fraction in contrast to undetectable levels of A20.1-mFc after a 5-min perfusion protocol. Systemic administration of IGF1R4-mFc fused with the non-BBB crossing analgesic peptide galanin (2 and 5 mg/kg) induced a dose-dependent suppression of thermal hyperalgesia in the Hargreaves pain model. In conclusion, novel anti-IGF1R sdAbs showed receptor-mediated brain uptake with pharmacologically effective parenchymal delivery of non-permeable neuroactive peptides.

Development of a Blood-Brain Barrier Permeability Assay Using Human Induced Pluripotent Stem Cell Derived Brain Endothelial Cells.

The development of translational and predictive models in vitro for assessing blood-brain barrier (BBB) delivery has become an important requirement in preclinical testing of CNS-targeting therapeutics. Here we describe a directed monolayer differentiation strategy to generate a population of brain endothelial-like cells (BECs) from human induced pluripotent stem cell (iPSC) with robust BBB properties. To generate BBB permeability assays, the BECs are seeded as a monolayer on a semipermeable Transwell insert placed inside a companion plate to generate a two-compartment Transwell model. The BECs provide a BBB-like separation between the luminal (blood) and abluminal (brain) compartments to assess BBB permeability of CNS-targeting therapeutics.

Proteome of the Luminal Surface of the Blood-Brain Barrier.

Interrogation of the molecular makeup of the blood-brain barrier (BBB) using proteomic techniques has contributed to the cataloguing and functional understanding of the proteins uniquely organized at this specialized interface. The majority of proteomic studies have focused on cellular components of the BBB, including cultured brain endothelial cells (BEC). Detailed proteome mapping of polarized BEC membranes and their intracellular endosomal compartments has led to an improved understanding of the processes leading to internalization and transport of various classes of molecules across the BBB. Quantitative proteomic methods have further enabled absolute and comparative quantification of key BBB transporters and receptors in isolated BEC and microvessels from various species. However, translational studies further require in vivo/in situ analyses of the proteins exposed on the luminal surface of BEC in vessels under various disease and treatment conditions. In vivo proteomics approaches, both profiling and quantitative, usually rely on 'capturing' luminally-exposed proteins after perfusion with chemical labeling reagents, followed by analysis with various mass spectrometry-based approaches. This manuscript reviews recent advances in proteomic analyses of luminal membranes of BEC in vitro and in vivo and their applications in translational studies focused on developing novel delivery methods across the BBB.

Differentiation of Adipose-Derived Stem Cells into Vascular Smooth Muscle Cells for Tissue Engineering Applications.

Synthetic grafts have been developed for vascular bypass surgery, however, the risks of thrombosis and neointimal hyperplasia still limit their use. Tissue engineering with the use of adipose-derived stem cells (ASCs) has shown promise in addressing these limitations. Here we further characterized and optimized the ASC differentiation into smooth muscle cells (VSMCs) induced by TGF-ß and BMP-4. TGF-ß and BMP-4 induced a time-dependent expression of SMC markers in ASC. Shortening the differentiation period from 7 to 4 days did not impair the functional property of contraction in these cells. Stability of the process was demonstrated by switching cells to regular growth media for up to 14 days. The role of IGFBP7, a downstream effector of TGF-ß, was also examined. Finally, topographic and surface patterning of a substrate is recognized as a powerful tool for regulating cell differentiation. Here we provide evidence that a non-woven PET structure does not affect the differentiation of ASC. Taken together, our results indicate that VSMCs differentiated from ASCs are a suitable candidate to populate a PET-based vascular scaffolds. By employing an autologous source of cells we provide a novel alternative to address major issues that reduces long-term patency of currently vascular grafts.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human.

Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood-brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. Constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of antibodies or protein ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter SLC3A2/CD98hc and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, brain parenchyma and peripheral organs of the mouse and the human using RNA-seq approach. IGF1R, INSR and LRP8 were highly enriched in mouse brain microvessels compared to peripheral tissues. In human brain microvessels only INSR was enriched compared to either the brain or the lung. The expression levels of SLC2A1, LRP1, IGF1R, LRP8 and TFRC were significantly higher in the mouse compared to human brain microvessels. The protein expression of these receptors analyzed by Western blot and immunofluorescent staining of the brain microvessels correlated with their transcript abundance. This study provides a molecular transcriptomics map of key RMT receptors in mouse and human brain microvessels and peripheral tissues, important to translational studies of biodistribution, efficacy and safety of antibodies developed against these receptors.

In vivo near-infrared fluorescent optical imaging for CNS drug discovery.

INTRODUCTION: In vivo imaging technologies have become integral and essential component of drug discovery, development, and clinical assessment for central nervous system (CNS) diseases. Near-infrared (NIR) fluorescence imaging in the range of 650-950 nm is widely used for pre-clinical in vivo imaging studies. The recent expansion of NIR imaging into the shortwave infrared (SWIR, 1000-1700 nm) window enabled improvements in tissue penetration and resolution required for anatomical, dynamic, and molecular neuroimaging with high potential for clinical translation. AREAS COVERED: This review focuses on the latest progress in near-infrared (NIR)-fluorescent optical imaging modalities with an emphasis on the SWIR window. Advantages and challenges in developing novel organic and inorganic SWIR emitters, with special attention to their toxicology and pharmacology, are discussed. Examples of their application in preclinical imaging of brain function and pathology provide a platform to assess the potential for their clinical translation. EXPERT OPINION: Propelled through concomitant technological advancements in imaging instrumentation, algorithms and new SWIR emitters, SWIR imaging has addressed key barriers to optical imaging modalities used in pre-clinical studies addressing the CNS. Development of biocompatible SWIR emitters and adoption of SWIR into multi-modal imaging modalities promise to rapidly advance optical imaging into translational studies and clinical applications.

Prioritization of Therapeutic Targets of Inflammation Using Proteomics, Bioinformatics, and In Silico Cell-Cell Interactomics.

Protein-protein interactions play key roles in leukocyte extravasation process into the brain and have been attractive therapeutic targets for inhibiting brain inflammation using blocking (or neutralizing) antibodies. These targets include protein-protein interactions between cytokines (or chemokines) and their receptors on leukocytes and between adhesion molecules of leukocyte and brain endothelium. While a number of therapeutics against these targets are currently used in clinic for treatment of brain autoimmune and inflammatory disorders (e.g., multiple sclerosis), they are associated with side effects partly due to the off-target actions (i.e., nonspecific targets). There is a need for novel targets involved in the leukocyte extravasation process that are specific to leukocyte subsets or to individual inflammatory disorder and are amenable for drug development (i.e., druggable). We recently described the blood-brain barrier (BBB) Carta Project as a comprehensive collection of molecular "maps" consisting of multiple experimental omics (including RNA sequencing, proteomics, glycoproteomics, glycomics, metabolomics) and in silico informatics analyses on a number of mammalian species from hundreds of internal, publically available, or curated datasets. Utilizing the datasets and tools from the BBB Carta Project, we describe a methodology to identify novel "druggable" targets involving protein-protein interactions between activated leukocytes and brain endothelial cells using a combination of proteomics, bioinformatics, and in silico interactomics. The result is a prioritized list of protein-protein interactions in a network consisting of leukocyte-brain endothelial cell communication and contacts. These interactions can be further pursued for development of therapeutics such as neutralizing antibodies and their validation through preclinical testing. In addition to targeting brain inflammation, the method described here is applicable for peripheral inflammation and provides the opportunity to target important cell-cell interactions and communications that are more specific/selective for inflammatory disorders and improve currently available therapies.

Emerging Technologies for Delivery of Biotherapeutics and Gene Therapy Across the Blood-Brain Barrier.

Antibody, immuno- and gene therapies developed for neurological indications face a delivery challenge posed by various anatomical and physiological barriers within the central nervous system (CNS); most notably, the blood-brain barrier (BBB). Emerging delivery technologies for biotherapeutics have focused on trans-cellular pathways across the BBB utilizing receptor-mediated transcytosis (RMT). 'Traditionally' targeted RMT receptors, transferrin receptor (TfR) and insulin receptor (IR), are ubiquitously expressed and pose numerous translational challenges during development, including species differences and safety risks. Recent advances in antibody engineering technologies and discoveries of RMT targets and BBB-crossing antibodies that are more BBB-selective have combined to create a new preclinical pipeline of BBB-crossing biotherapeutics with improved efficacy and safety. Novel BBB-selective RMT targets and carrier antibodies have exposed additional opportunities for re-targeting gene delivery vectors or nanocarriers for more efficient brain delivery. Emergence and refinement of core technologies of genetic engineering and editing as well as biomanufacturing of viral vectors and cell-derived products have de-risked the path to the development of systemic gene therapy approaches for the CNS. In particular, brain-tropic viral vectors and extracellular vesicles have recently expanded the repertoire of brain delivery strategies for biotherapeutics. Whereas protein biotherapeutics and bispecific antibodies enabled for BBB transcytosis are rapidly heading towards clinical trials, systemic gene therapy approaches for CNS will likely remain in research phase for the foreseeable future. The promise and limitations of these emerging cross-BBB delivery technologies are further discussed in this article.

Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood-brain barrier in vitro is dependent on its binding affinity.

The blood-brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re-directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter-study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (Kd from 5 to174 nM) of the rat TfR-binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics-selected reaction monitoring and fluorescent imaging with markers of early- and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co-localized with late-endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co-localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.

Zika virus crosses an in vitro human blood brain barrier model.

Zika virus (ZIKV) is a flavivirus that is highly neurotropic causing congenital abnormalities and neurological damage to the central nervous systems (CNS). In this study, we used a human induced pluripotent stem cell (iPSC)-derived blood brain barrier (BBB) model to demonstrate that ZIKV can infect brain endothelial cells (i-BECs) without compromising the BBB barrier integrity or permeability. Although no disruption to the BBB was observed post-infection, ZIKV particles were released on the abluminal side of the BBB model and infected underlying iPSC-derived neural progenitor cells (i-NPs). AXL, a putative ZIKV cellular entry receptor, was also highly expressed in ZIKV-susceptible i-BEC and i-NPs. This iPSC-derived BBB model can help elucidate the mechanism by which ZIKV can infect BECs, cross the BBB and gain access to the CNS.