Background
The receptor-mediated transcytosis (RMT) is a vesicular transcellular route by which various macromolecules are transported across a barrier, typically formed by a cell monolayer [
1,
2]. This process is particularly important for brain delivery of essential macromolecules, including transferrin and insulin [
3‐
6], across the blood–brain barrier (BBB) [
1,
2]. A ligand binding to a receptor on the luminal surface of brain endothelial cells (BEC) triggers ligand-receptor complex endocytosis, routing through various intracellular endosomal compartments where cargo is detached from the receptor and released on the abluminal side, while the receptor recycles ‘back’ to accept additional cargo molecules [
7‐
11]. This pathway has been particularly well described for the transferrin receptor (TfR), which undergoes constitutive recycling and has been considered a ‘prototypical’ trigger of the RMT pathway [
7‐
13].
Antibodies and peptide ligands against various endocytosing BBB receptors have been developed as potential molecular Trojan horses or shuttles to deliver therapeutic cargos across the BBB [
2,
8‐
16]. Discovery strategies utilizing molecular (‘omics’) analyses of the BBB and screening of antibody libraries to select antibody species that can transmigrate the BBB [
17‐
22] have yielded an experimental proof of concept for novel RMT-antibody pairs. The literature survey of BBB targets shown to mediate transcytosis of antibodies or protein ligands in various model systems in vitro and in vivo, identified the following broad classes of transporters: (a)
iron transporters: transferrin receptor (TFRC) [
3‐
6,
8,
12,
13]; (b)
insulin and insulin-
like growth factors receptors: INSR, IGF1R, and IGF2R [
16,
23,
24]; (c)
lipid transporters: low-density lipoprotein receptor (LDLR), LDLR-related protein 1 (LRP1), LRP8, and transmembrane protein 30A (TMEM30A/CDC50A) [
25‐
32]; (d)
solute carrier family transporters: glucose transporter-1 (GLUT-1)/SLC2A1 and SLC3A2/CD98hc [
33,
34]; and e)
neuropeptide receptors: leptin receptor (LEPR) [
35]. Structure/function relationships, ligand interactions and signaling, and specialized biology of these receptors is vastly different (summarized briefly in Additional file
1: Table S1), expanding the spectrum of potential RMT pathways across the BBB beyond the ‘canonical’ mechanism of TfR recycling. Whereas the mechanisms of antibody transport by some of these targets, in particular SLC family of transporters, is not fully understood, for the purpose of this manuscript they will be considered in comparison with ‘classical’ RMT-triggered pathways.
The development of peptides and antibodies that bind RMT receptors as potential delivery carriers for brain-targeting biotherapeutics brought about further understanding of RMT mechanisms and underscored key limitations of various approaches. For example, evaluation of bi-specific antibodies where the TfR antibody was used as a BBB transport shuttle [
36,
37] revealed TfR antibody-driven fast systemic clearance, on-target complement-mediated toxicity on TfR-rich reticulocytes, as well as species differences in transport capacity in mice and non-human primates [
7,
36,
38]. Ideally, RMT receptors targeted for the development of BBB delivery antibodies should demonstrate a high degree of BBB expression selectivity compared to peripheral organs, a high abundance in the brain endothelium, as well as low expression in the brain parenchyma [
8,
9]. This profile would enable an extended systemic half-life and reduced toxicity, high efficiency of brain delivery and low target-mediated removal in the brain tissue. For translational preclinical studies, and, ultimately, for demonstration of the concept in human patients, it is also important to consider species differences in the RMT target expression [
39]. Three BBB carriers targeting RMT pathway, a peptide ligand of LRP-1 (Angiopep) [
26,
27,
40], anti-human INSR antibody [
14,
15,
41], and anti-TfR antibody [
42] are being tested in clinical trials; however, the clinical proof of concept for RMT pathway as a viable route for systemic delivery of CNS biotherapeutics remains inconclusive.
The principal objective of the current study was to compare the transcript abundance of selected receptors and transporters previously shown to mediate transport of antibodies or protein ligands into the brain in isolated brain microvessels from mouse, the principal experimental species used in pre-clinical studies, and from human, by using RNA-seq. In addition, transcript abundance for these receptors was determined in the whole brain tissue and in various peripheral organs. Significant species differences in RMT receptor abundances and distribution among brain microvessels, brain and peripheral organs were observed between mouse and human. This study suggests that the RMT target receptor abundance is an important parameter to consider in translational studies for antibody-based BBB delivery strategies.
Materials and methods
Human and mouse tissues
Samples of the human brain and lung tissues from three individuals were used in the study. Human lung tissues were from normal adjacent lung tissues of three non- small cell lung cancer (NSCLC) patients undergoing surgical resections. The surgical samples were deposited in the Lung Tumor Bank managed by the CDHA-Capital District Health Authority at Halifax, NS, Canada. All patients signed informed consent as per CDHA-RS/2013-271, which allowed their tissues to be archived in the CDHA Lung Tumor Bank for molecular studies. Human post mortem brain tissues (two females and one male, 73–76 years old; deceased from non-brain related pathologies) were obtained from the Human Brain and Spinal Fluid Resource Center, VAMC (Los Angeles, CA), which is sponsored by NINDS/NIMN, National Multiple Sclerosis Society, VA Greater Los Angeles Healthcare System, and Veterans Health Services and Research Administration, Department of Veteran Affairs. The use of human tissues in this study was approved by the Research Ethics Board of the National Research Council Canada (Protocols #2013-38 and #2006-03). The use of mice in this study was approved by the Animal Care Committee of the Human Health Therapeutics Research Centre at the National Research Council of Canada (Animal User Protocol #2016-04). Mice (C57BL/6 J strain, three males, 9-month old) were perfused transcardially with heparnized saline (15 mL at 2 mL/min) and organs were collected. Brain and brain vessels were freshly isolated without freezing; other organs were frozen at − 80 °C until RNA isolation.
Isolation of microvessels and capillaries from tissues
Microvessels and capillaries were isolated from mouse and human brains, as well as mouse lungs using modified protocols described previously [
43]. Mouse brain and lung vessels were isolated from fresh tissues, whereas all human tissues were frozen and stored at − 80 °C until vessel isolation. The tissues were pre-weighed and thawed briefly at room temperature. Tissue homogenization and vessel separation were performed on ice using PBS (Wisent, St-Bruno, QC) containing protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO.) or using a buffer containing 150 mM NaCl, 50 mM Tris (BDH, Ward Chester, PA) pH 8.0 for mass spectrometry analysis with instruments pre-chilled on ice. Respective tissues were chopped using razor blade, placed in a 5-mL Wheaton Dounce homogenization tube (Fisher Scientific, Hampton, NH), and 5 mL of PBS buffer was added per tube. Tissue was homogenized with 10 strokes of the pestle connected to Eberbach Con-Torque Homogenizer (Fisher Scientific, Hampton, NH). The tissue homogenate was transferred onto pluriStrainers in a 50-mL conical tube with a connector ring and strainers (pluriSelect, San Diego, CA) in descending order, as follows: 300 µm, 100 µm, and 20 µm strainers and connector ring, and then gentle suction was used to filter the homogenate through strainers.
Stacked strainers were rinsed with 5–10 mL of PBS and vessels were collected by placing strainers upside down in a new 50-mL conical tube followed by a rinse with 5-mL PBS per each strainer. To release the microvessels and capillaries from the strainer, the buffer was forced through the filter by pipetting up and down with a P1000 pipette. Vessels collected onto 100 μm and 20 μm strainers were collected in the same 50-mL conical tube and were centrifuged at 900g for 5 min at 4 °C to pellet the vessels. The supernatant was carefully aspirated, and vascular pellets, designated as brain microvessels (BMV) were then processed for proteomics, immunofluorescence and RNA extraction. Vessel-depleted brain filtrates were also collected to analyze protein expression in brain parenchyma.
RNA isolation
RNA was extracted from isolated BMVs by using RNeasy Plus Mini kit (Qiagen, Toronto, ON), while NucleoSpin RNA plus kit (Macherey–Nagel GmbH & Co. KG) was used for RNA isolation from all other tissues following manufacturer’s protocols. Genomic DNA contamination was removed by Turbo DNA-Free Kit (Life Technologies/ThermoFisher Scientific, Nepean, ON). RNA quality was assessed using Agilent Bioanalyzer 2100 (Santa Clara, CA).
RNA-seq
RNA-Seq Libraries were generated using the TruSeq strand RNA kit (Illumina, San Diego, CA). The RNA-Seq libraries were quantified by Qbit and qPCR according to the Illumina Sequencing Library qPCR Quantification Guide and the quality of the libraries was evaluated on Agilent Bioanalyzer 2100 using the Agilent DNA-100 chip (Santa Clara, CA). The RNA-Seq library sequencing was performed using Illumina Next-Seq 500. FASTQ file format was processed by trimming the adaptor sequences, filtering low-quality reads (Phred Score ≤ 20) and eliminating short reads (length ≤ 20 bps) using software package FASTX-toolkit [
http://hannonlab.cshl.edu/fastx_toolkit/]. STAR (v2.5.3a) [
44] was used for the alignment of reads to the reference genome and to generate gene-level read counts. RSEM (version 1.3.3) [
45] was used for alignment, to generate Transcripts per million (TPM) count. Mouse reference genome (version GRCm38.p6, M24), human reference genome (version GRCh38.p13, Genecode 33) and corresponding annotations were used as references for RNA-seq data alignment process. DESeq 2 [
46] was used for data normalization and differentially expressed gene identification for each pair-wise comparison.
Public data sets and analysis
RNA-seq and microarray data in the public domains were obtained to compare/benchmark the data generated from this study for quality and comparability purposes. For RNA-seq data, raw data corresponding to normal lung and brain samples were obtained from the Sequence Read Archive [
47] from the Genomics Data Commons [
48]. GTEx data were processed using GDC reference files using GDC mRNA analysis pipeline (STAR two-pass) [
44]. These data were combined with 12 samples analyzed at NRC and processed using DESeq 2 [
46].
Automated Western blot analysis (Wes™)
Human and mouse BMVs were lysed in Cellytic MT buffer (Sigma) with 1 X Complete protease inhibitor (Roche) pellets on ice. The lysates were incubated on ice for 30 min, vortexed, then centrifuged at 21,000×g for 10 min in a Sorvall Legend Micro 21R centrifuge. Protein concentrations were determined using the Quantipro BCA Assay Kit (Sigma). Wes was run using the 12–230kDA separation module (ProteinSimple), and the mouse or rabbit detection module (ProteinSimple Inc., San Jose, CA). Wes samples (protein at 0.8 mg/mL) were prepared by combining Master Mix to sample in a 1:4 ratio. Samples and Biotinylated Ladder were heated in a Accublock digital dry bath at 95 °C for 5 min. Samples were cooled to room temperature, vortexed to mix and centrifuged in a Mandel mini microfuge. Biotinylated ladder, samples, primary and secondary antibodies, and luminol were loaded on the plate and Wes was run using the standard protocol. Primary antibodies were rabbit anti-IGF1 receptor β (Cell Signaling, 3027S), mouse anti-transferrin receptor (Invitrogen, 13-6800) and rabbit anti-LRP1 (Abcam, ab925443). Primary antibodies were cross-reactive with human and mouse IGF1R, LRP1 and TfR proteins. Streptavidin-HRP was used to detect the ladder proteins. Data for each sample was first normalized to β-actin in the same lane. The level of the protein in mouse BMV was set as one fold. The fold-change of human protein was calculated relative to mouse protein (Mean ± SD).
Immunofluorescence
Isolated brain microvessels in PBS (5 µL) were deposited on Superfrost Plus slides (Fisher Scientific, Toronto, ON) and air-dried for 30 min. Samples were then fixed in Methanol (Fisher Scientific, Toronto, ON) for 15 min at room temperature and followed by incubation in 5% Normal Goat or Normal Donkey Serum (Jackson ImmunoResearch, West Grove, PA) in 0.2% Triton X-100 (Sigma, Oakville, ON) solution for 1 h at room temperature. The vessels were incubated with CD31 antibody 1:300 (BD Biosciences, San Jose, CA), Collagen IV antibody 1:300 (Millipore, Etobicoke, ON) or Griffonia Simplicifolia Lectin I (GSL-I) 1:500 (Vector Labs, Burlingame, CA). The primary antibodies for TfR, LRP-1 and IGF1R are the same as used above for Wes™ analyses. Secondary antibodies used were Goat anti-Rat and Donkey anti-Goat Alexa conjugated (Fisher Scientific, Toronto, ON) at 1:500 dilutions for 1 h at room temperature. Samples were mounted in Fluorescent Mounting Medium (Dako, Santa Clara, CA) Containing 2 µg/mL Hoechst (Fisher Scientific, Toronto, ON). Images were acquired using Zeiss Axiovert 200 M microscope with 20× objective.
Tissue-Immunocytochemistry
Isolated brain vessels were air-dried for 15 min on slide warmer at 37 °C and fixed using commercially available Zinc 10× fixative (diluted to 1x with MQH2O) for 90 min at RT. The slides were rinsed 3 times with PBS containing 0.05% tween-20 and blocked with Dako serum-free protein block containing 0.1% Triton-X 100 for 1 h at room temperature (RT). Endogenous peroxidase was quenched with 3% H2O2 in PBS for 15 min RT. The slides were rinsed 3 times with PBS containing 0.05% tween-20. IGF1R antibody (at 1:100 dilution; Cell Signaling 3027S lot13) was applied to the slides for 2 h RT (diluted in Dako antibody diluent containing 0.05% Triton-X 100). The slides were washed 3 times with PBS containing 0.05% tween-20 (at 2 min each). Secondary biotinylated goat anti-rabbit antibody was then applied to the slides for 45 min RT (1:250, diluted in Dako antibody diluent). The slides were washed 3 times with PBS containing 0.05% tween-20 (at 2 min each). RTU Horseradish peroxidase Avidin D was then applied for 30 min at RT. The slides were washed 3 times with PBS containing 0.05% tween-20 (at 2 min each). Color was developed 8 min with DAB substrate kit Vector labs SK4100. Slides were counterstained using hematoxylin, then dehydrated, cleared and cover slipped with xylene-based mounting media. The slides were visualized by Olympus IX81 inverted microscopy (Richmond Hill, ON). Images were acquired using Olympus IX81 inverted microscopy with 20× objective.
Nano LC–MS/MS analysis
Peptides from digested proteins prepared as described [
10] were analyzed on a reversed-phase nanoAcquity UPLC (Waters, Milford, MA) coupled with a LTQ-XL mass spectrometer (ThermoFisher, Waltham, MA) with a nano-electrospray interface operated in positive ion mode. The analysis involved injection and loading of 0.6–1 μg of mouse brain protein onto a 300 µm I.D. × 0.5 mm 3 µm PepMaps
® C18 trap (ThermoFisher) followed by eluting onto a 100 µm I.D. × 10 cm 1.7 µm BEH130C18 nanoLC column (Waters). The mobile phase consisted of 0.1% (v/v) formic acid in water as buffer A and 0.1% (v/v) formic acid in acetonitrile as buffer B. The peptides were separated using a gradient ramping from 0.1 to 45% buffer B over 40 min, 45%–85% buffer B over 2 min, and then re-equilibrating from 85 to 0.1% buffer B over 10 min at a flow rate of 600 nL/min. The eluted peptides were ionized by electrospray ionization (ESI). Data were acquired on ions with mass/charge (m/z) values between 350 and 2000 followed by three data-dependent MS/MS scans using collision-induced dissociation (CID) for fragmentation of the peptide ions.
Statistical analysis
The data from multiple groups were analyzed and compared using One-way ANOVA followed by Tukey’s multiple comparisons test. Unpaired two-tailed student t test was used to compare data between two groups. p < 0.05 is considered statistical significance.
Discussion
Antibodies or peptide ligands against brain endothelial cell receptors that undergo RMT have been developed for brain delivery of therapeutic cargos. This strategy exploits a natural transport function of these receptors, which supply the brain with essential proteins (such as transferrin, insulin and insulin-like growth factors), peptides (such as leptin), essential amino acids, glucose or lipids. However, most of these receptors also play important functions and are expressed broadly or selectively in peripheral organs and tissues. In addition, their expression levels and distribution may vary in different species or pathologies, confounding translational development of BBB delivery carriers. The expression levels of genes encoding RMT receptors in different tissues and species have not been systemically investigated.
The selection of RMT receptors or other transporters for the development of BBB shuttles (carriers) should be guided by several criteria, including a) the selectivity of the expression in BEC compared to peripheral tissues and organs, to assure good systemic pharmacokinetics and to mitigate potential toxicity; b) low expression in brain parenchymal cells to avoid off-target distribution of the cargo, especially when cargo is an antibody against parenchymal target; c) abundance of the receptor in BEC to assure a sufficient transport capacity for a given application; c) similar patterns of expression in both BEC and other organs in experimental pre-clinical species and in humans to ensure translation of experimental findings to clinical application; d) the ability of ligands (natural or engineered) to the receptor to internalize into and to transcytoses/release on the abluminal side of the brain endothelial cells.
To facilitate translational studies of BBB RMT carriers (shuttles) developed against various RMT receptors described in the literature (Additional file
1: Table S1), we have compared their abundance and enrichment in isolated mouse or human brain microvessels (BMVs) to those in either the whole brain or peripheral tissues. The study revealed a significant species differences in selected RMT receptor abundance in different organs, as well as in BMVs.
The case study of TfR antibodies illustrates important translational hurdles in the pre-clinical to clinical development path of BBB carrier antibodies. TfR antibodies with various characteristics have been developed for delivery of biotherapeutic cargos across the BBB, notably TfR/BACE1 bispecific antibody [
7] and an anti-Aβ antibody fused to a single TfR-binding Fab fragment [
37]. TfR is highly expressed in reticulocytes causing on-target toxicity of effector-function competent antibodies [
38]; high-affinity TfR antibodies also exhibit poor pharmacokinetics due to peripheral target-mediated clearance [
42,
53]. Due to lack of species cross-reactivity of TfR antibodies, the development of transgenic mouse expressing human TfR extracellular domain [
7,
12,
13,
37], use of surrogate antibody or of transgenic mouse expressing human TfR [
14,
15,
37] were necessary in pre-clinical studies. The translational PK-PD models developed based on these studies [
54] did not take into account differences in TfR abundance between mouse and human. Comparative analyses of the TFRC expression performed in this study demonstrated a significantly higher abundance of TFRC in the mouse compared to human BMVs. In both human and mouse BMVs, TfR abundance was lower than those of SLC3A2/CD98hc and LRP1. Clinical trials with TfR antibodies as BBB carriers have been initiated by Roche (to deliver Aβ-antibody for treatment of Alzheimer’s disease) and by JCR Pharma (to deliver iduronate-2-sulfatase enzyme for treatment of Mucopolysaccharidosis II) [
42]. These studies will provide further understanding of how differential TfR abundance in the brain vasculature, the brain and peripheral tissues between pre-clinical mouse species and humans may influence pharmacokinetics, toxicity and brain exposure of TfR antibody-containing biologics tested in the clinic.
Our study further found that: 1) INSR, IGF1R, LRP1 and LRP8 were enriched in the mouse BMVs compared to peripheral tissues; and INSR was enriched in human BMVs compared to total brain and lung; 2) SLC2A1/GLUT1, LRP-1, SLC3A2/CD98hc and CDC50A were the most abundant among receptor/transporters studied in human BMVs; whereas SLC2A1/GLUT1, LRP-1, SLC3A2/CD98hc and IGF1R were the most abundant in mouse BMVs; 3) mouse BMVs have significantly higher abundance of TfR, INSR, IGF1R, LRP-1, LRP8 and CDC50A compared to human BMVs.
The caveat of the current study is that RMT gene transcripts were analyzed in isolated BMVs which contain more than one cell type. A strong endothelial enrichment in our BMV preparations has been confirmed by RNA-seq, targeted proteomics and immunofluorescence analyses; however, the presence of contaminating pericytes and astrocytic end-feet still confound the interpretation of the results. Therefore it is important to interpret results from this study in conjunction with the data obtained by other published studies that analyzed transcript profiles in brain cells or vascular segments using single-cell sequencing [
20,
49,
52]. Human BMVs from this study showed comparatively lower expression of TfR, IGF1R, LRP8, LDLR and LEPR from that found in BEC obtained by the single-cell sequencing of human brain [
20]. While transcript dilution as a result of the presence of multiple cell types, notably pericytes, in microvessel preparations compared to the purified cells, the other factors such as age of the donors, could also contribute to this observation. In contrast, RMT receptor expression in mouse BMVs was comparatively similar to that observed in BEC by the single-cell sequencing of mouse brain vessels [
19], with the exception of the higher expression of LRP1 in isolated BMVs. Single-cell sequencing studies of human and mouse brain and brain vessels [
19,
21] both found LRP-1 expression to be very low in BEC, in contrast to high expression in brain vascular smooth muscle cells, pericytes and fibroblast-like cells. A study in cultured bovine NVU cells showed eightfold higher expression of LRP-1 in pericytes compared to brain endothelial cells [
55]. This suggests that, despite being among the most abundant transcripts in isolated BMVs, LRP-1 expression originates from non-endothelial cells. This also brings into question its postulated role as BEC RMT receptor useful for therapeutic delivery across the BBB; it should be noted that antibodies raised against LRP1 failed to show facilitated BBB crossing [
34].
Using proteomic survey, Zuchero et al. [
34] found a very high abundance of basigin (BSG), followed by TfR and SLC3A2/CD98hc, whereas LRP1 was expressed at very low levels in freshly isolated endothelial cells from the mouse brain. RNA-seq data of mouse BMVs in this study ranked the abundance of SLC3A2/CD98hc higher than that of TfR, and similar to that of IGF1R. SLC3A2/CD98hc was also strongly expressed in the mouse spleen, consistent with the reported high expression levels in lymphocytes [
56]. In contrast, SLC3A2/CD98hc expression was low in both human and mouse whole brain tissues.
Among the putative RMT receptors analyzed, SLC2A1 and IGF1R showed the highest selectivity of expression in mouse BMVs compared to all peripheral tissues, as well as the whole brain. IGF1R transcript levels were twofold more abundant than the TfR in mouse BMVs and were similar to that of the TfR in human BMVs. A recent single-cell transcriptomics study of endothelial cells in various mouse vascular beds [
52], also found IGF1R highly enriched in BEC and threefold more abundant than TfR. In contrast, IGF2R exhibited low abundance in BMVs and high levels in lung tissues. This is consistent with the known developmental down-regulation of IGF2R in adult BBB, also accompanied by the lost ability of the receptor to facilitate transport of mannose 6-phosphate enriched lysosomal storage enzymes into the brain [
57].
The additional caveat of transcriptomics studies is that the protein expression/function does not always correlate with the transcript abundance in given tissues. For example, a quantitative proteomics study of transporters and receptor in isolated human BMVs observed higher expression of TfR (2.34 ± 0.76 fmol/µg protein) compared to either LRP-1 (1.51 ± 0.26 fmol/µg protein) or INSR (1.09 ± 0.21 fmol/µg protein) [
58], whereas in this study the LRP-1 transcript abundance in human BMVs was the highest among studied putative RMT receptors; albeit likely due to its enrichment in pericytes. Our within-study comparisons between the transcript abundance and the protein expression by Wes analysis for TfR, LRP-1 and IGF1R showed similar trends of lower expression of IGF1R and TfR in human compared to mouse BMVs; whereas no difference was found for LRP1. A species cross-reactive antibodies used in these studies make true quantification of expression levels difficult; for example immunodetection studies with the same antibodies did not detect appreciable expression differences between human and mouse BMVs, likely because antigen retrieval and antibody reactivity to antigenic epitopes in two methods are very different.
Conclusion
This study provides a molecular transcriptomics map of key RMT receptors in mouse and human brain microvessels, brain and peripheral tissues, important to translational studies of biodistribution, efficacy and safety of antibodies developed against these receptors.
RMT receptor genes in brain microvessels, brain parenchyma and peripheral organs from human and mouse were analyzed by RNA-seq and validated by Western blot and in situ immunodetection. SLC2A1, IGF1R, INSR and LRP8 were highly enriched in mouse BMVs compared to peripheral tissues; additionally SLC2A1 and IGF1R showed high abundance in mouse BMVs. Antibodies against IGF1R have been shown to internalize into brain endothelial cells; while antibodies against both these targets (SLC2A1 and IGF1R) showed enhanced total brain levels in mice compared to control antibodies (although in the absence of vessel depletion for SLC2A1 antibodies). Among evaluated receptors, IGF1R and SLC2A1 appear to satisfy most of the criteria for promising targets to develop trans-BBB delivery ligands; however, it should be understood that SLC2A1 is a carrier-mediated transporter unlikely to undergo a typical RMT. In addition to desired characteristics of the target receptor, development and optimization of an optimized ligand/antibody against these targets is essential for achieving success in improved delivery of biotherapeutics across the BBB.
Cross-species differences in the expression of RMT in BMVs, brain and peripheral tissues is an important consideration in translational studies to advance BBB carriers developed against these receptors into clinical trials. This study suggests that the mouse BBB expresses many of targeted RMT receptors at higher levels than the human BBB. Since the mouse is the most widely used pre-clinical model for discovery and evaluation of investigational compounds, including brain delivery ‘shuttles’, translational studies and PK-PD modeling to estimate dosing in humans need to account for species differences in RMT receptor abundance in BEC and different organs.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.