Abstract
The blood-brain barrier (BBB) is a major obstacle for the treatment of central nervous system (CNS) disorders. Significant progress has been made in developing adeno-associated virus (AAV) variants with increased ability to cross the BBB in mice. However, these variants are not efficacious in non-human primates. Herein, we employed various bioinformatic techniques to identify lymphocyte antigen-6E (LY6E) as a candidate for mediating transport of AAV across the human BBB based on the previously determined mechanism of transport in mice. Our results provide insight into future discovery and optimization of AAV variants for CNS gene delivery in humans.
This is a preview of subscription content, log in via an institution to check access.
References
Agbandje-McKenna M, Kleinschmidt J (2011) AAV capsid structure and cell interactions. Methods Mol Biol (Clifton, NJ) 807:47–92. https://doi.org/10.1007/978-1-61779-370-7_3
Aponte-Ubillus JJ, Barajas D, Peltier J, Bardliving C, Shamlou P, Gold D (2018) Molecular design for recombinant adeno-associated virus (rAAV) vector production. Appl Microbiol Biotechnol 102(3):1045–1054. https://doi.org/10.1007/s00253-017-8670-1
Argyris EG, Acheampong E, Nunnari G, Mukhtar M, Williams KJ, Pomerantz RJ (2003) Human immunodeficiency virus type 1 enters primary human brain microvascular endothelial cells by a mechanism involving cell surface proteoglycans independent of lipid rafts. J Virol 77(22):12140–12151. https://doi.org/10.1128/jvi.77.22.12140-12151.2003
Berndt P, Winkler L, Cording J, Breitkreuz-Korff O, Rex A, Dithmer S, Rausch V, Blasig R, Richter M, Sporbert A, Wolburg H, Blasig IE, Haseloff RF (2019) Tight junction proteins at the blood–brain barrier: far more than claudin-5. Cell Mol Life Sci 76(10):1987–2002. https://doi.org/10.1007/s00018-019-03030-7
Bobardt MD, Salmon P, Wang L, Esko JD, Gabuzda D, Fiala M, Trono D, Van der Schueren B, David G, Gallay PA (2004) Contribution of proteoglycans to human immunodeficiency virus type 1 brain invasion. J Virol 78(12):6567–6584. https://doi.org/10.1128/JVI.78.12.6567-6584.2004
Büning H, Schmidt M (2015) Adeno-associated vector toxicity—to be or not to be? Mol Ther 23(11):1673–1675. https://doi.org/10.1038/mt.2015.182
Büning H, Srivastava A (2019) Capsid modifications for targeting and improving the efficacy of AAV vectors. Mol Therapy Methods Clin Dev 12:248–265. https://doi.org/10.1016/j.omtm.2019.01.008
Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, Reier PJ, Mandel RJ, Muzyczka N (2004) Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol Therapy 10(2):302–317. https://doi.org/10.1016/j.ymthe.2004.05.024
Chan KY, Jang MJ, Yoo BB, Greenbaum A, Ravi N, Wu W-L, Sánchez-Guardado L, Lois C, Mazmanian SK, Deverman BE, Gradinaru V (2017) Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci 20(8):1172–1179. https://doi.org/10.1038/nn.4593
Dashkoff J, Lerner EP, Truong N, Klickstein JA, Fan Z, Mu D, Maguire CA, Hyman BT, Hudry E (2016) Tailored transgene expression to specific cell types in the central nervous system after peripheral injection with AAV9. Mol Therapy Methods Clin Develop 3:16081. https://doi.org/10.1038/mtm.2016.81
Deverman BE, Pravdo PL, Simpson BP, Kumar SR, Chan KY, Banerjee A, Wu W-L, Yang B, Huber N, Pasca SP, Gradinaru V (2016) Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34(2):204–209. https://doi.org/10.1038/nbt.3440
Fagerberg L, Hallström BM, Oksvold P, Kampf C, Djureinovic D, Odeberg J, Habuka M, Tahmasebpoor S, Danielsson A, Edlund K, Asplund A, Sjöstedt E, Lundberg E, Szigyarto CA-K, Skogs M, Takanen JO, Berling H, Tegel H, Mulder J, Nilsson P, Schwenk JM, Lindskog C, Danielsson F, Mardinoglu A, Sivertsson Å, von Feilitzen K, Forsberg M, Zwahlen M, Olsson IM, Navani S, Huss M, Nielsen J, Ponten F, Uhlén M (2014) Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics 13(2):397–406. https://doi.org/10.1074/mcp.M113.035600
Fass D, Thorpe C (2018) Chemistry and enzymology of disulfide cross-linking in proteins. Chem Rev 118(3):1169–1198. https://doi.org/10.1021/acs.chemrev.7b00123
Ferreira V, Petry H, Salmon F (2014) Immune responses to AAV-vectors, the Glybera example from bench to bedside. Front Immunol 5. https://doi.org/10.3389/fimmu.2014.00082
Gaj T, Epstein BE, Schaffer DV (2016) Genome engineering using adeno-associated virus: basic and clinical research applications. Mol Ther 24(3):458–464. https://doi.org/10.1038/mt.2015.151
Go Y-M, Chandler JD, Jones DP (2015) The cysteine proteome. Free Radic Biol Med 84:227–245. https://doi.org/10.1016/j.freeradbiomed.2015.03.022
Goodall EF, Leach V, Wang C, Cooper-Knock J, Heath PR, Baker D, Drew DR, Saffrey MJ, Simpson JE, Romero IA, Wharton SB (2019) Age-associated mRNA and miRNA expression changes in the blood-brain barrier. Int J Mol Sci 20(12). https://doi.org/10.3390/ijms20123097
Gray, S. J., Choi, V. W., Asokan, A., Haberman, R. A., McCown, T. J., & Samulski, R. J. (2011). Production of recombinant adeno-associated viral vectors and use in in vitro and in vivo administration. Current Protocols in Neuroscience / Editorial Board, Jacqueline N. Crawley ... [et Al.], CHAPTER, Unit4.17. https://doi.org/10.1002/0471142301.ns0417s57
Hackett BA, Cherry S (2018) Flavivirus internalization is regulated by a size-dependent endocytic pathway. Proc Natl Acad Sci 115(16):4246–4251. https://doi.org/10.1073/pnas.1720032115
Haggerty DL, Grecco GG, Reeves KC, Atwood B (2020) Adeno-associated viral vectors in neuroscience research. Mol Ther Methods Clin Devel 17:69–82. https://doi.org/10.1016/j.omtm.2019.11.012
Hansen KD, Irizarry RA, Wu Z (2012) Removing technical variability in RNA-seq data using conditional quantile normalization. Biostatistics (Oxford, England) 13(2):204–216. https://doi.org/10.1093/biostatistics/kxr054
Hordeaux J, Wang Q, Katz N, Buza EL, Bell P, Wilson JM (2018) The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice. Mol Therapy 26(3):664–668. https://doi.org/10.1016/j.ymthe.2018.01.018
Hordeaux J, Yuan Y, Clark PM, Wang Q, Martino RA, Sims JJ, Bell P, Raymond A, Stanford WL, Wilson JM (2019) The GPI-linked protein LY6A drives AAV-PHP.B transport across the blood-brain barrier. Mol Therapy 27(5):912–921. https://doi.org/10.1016/j.ymthe.2019.02.013
Huang Q, Chan KY, Tobey IG, Chan YA, Poterba T, Boutros CL, Balazs AB, Daneman R, Bloom JM, Seed C, Deverman BE (2019) Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids. PLoS ONE 14(11):e0225206. https://doi.org/10.1371/journal.pone.0225206
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics (Oxford, England) 4(2):249–264. https://doi.org/10.1093/biostatistics/4.2.249
Jacus MO, Daryani VM, Harstead KE, Patel YT, Throm SL, Stewart CF (2016) Pharmacokinetic properties of anticancer agents for the treatment of central nervous system tumors: update of the literature. Clin Pharmacokinet 55(3):297–311. https://doi.org/10.1007/s40262-015-0319-6
Jamieson JJ, Searson PC, Gerecht S (2017) Engineering the human blood-brain barrier in vitro. J Biol Eng 11:37. https://doi.org/10.1186/s13036-017-0076-1
Kaisar MA, Sajja RK, Prasad S, Abhyankar VV, Liles T, Cucullo L (2017) New experimental models of the blood-brain barrier for CNS drug discovery. Expert Opin Drug Discovery 12(1):89–103. https://doi.org/10.1080/17460441.2017.1253676
Kalari KR, Thompson KJ, Nair AA, Tang X, Bockol MA, Jhawar N, Swaminathan SK, Lowe VJ, Kandimalla KK (2016) BBBomics-human blood brain barrier transcriptomics hub. Front Neurosci 10. https://doi.org/10.3389/fnins.2016.00071
Keeler AM, Flotte TR (2019) Recombinant adeno-associated virus gene therapy in light of Luxturna (and Zolgensma and Glybera): where are we, and how did we get here? Ann Rev Virol 6(1):601–621. https://doi.org/10.1146/annurev-virology-092818-015530
Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845–858. https://doi.org/10.1038/nprot.2015.053
Kong HK, Park JH (2012) Characterization and function of human Ly-6/uPAR molecules. BMB Rep 45(11):595–603. https://doi.org/10.5483/BMBRep.2012.45.11.210
Kurcinski M, Jamroz M, Blaszczyk M, Kolinski A, Kmiecik S (2015) CABS-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site. Nucleic Acids Res 43(W1):W419–W424. https://doi.org/10.1093/nar/gkv456
Leth JM, Leth-Espensen KZ, Kristensen KK, Kumari A, Lund Winther A-M, Young SG, Ploug M (2019) Evolution and medical significance of LU domain−containing proteins. Int J Mol Sci 20(11). https://doi.org/10.3390/ijms20112760
Loughner CL, Bruford EA, McAndrews MS, Delp EE, Swamynathan S, Swamynathan SK (2016) Organization, evolution and functions of the human and mouse Ly6/uPAR family genes. Human Genomics 10:10. https://doi.org/10.1186/s40246-016-0074-2
Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey ARN, Potter SC, Finn RD, Lopez R (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 47(W1):W636–W641. https://doi.org/10.1093/nar/gkz268
Manfredsson FP, Rising AC, Mandel RJ (2009) AAV9: a potential blood-brain barrier buster. Mol Therapy 17(3):403–405. https://doi.org/10.1038/mt.2009.15
Mann GE, Yudilevich DL, Sobrevia L (2003) Regulation of amino acid and glucose transporters in endothelial and smooth muscle cells. Physiol Rev 83(1):183–252. https://doi.org/10.1152/physrev.00022.2002
Mar KB, Rinkenberger NR, Boys IN, Eitson JL, McDougal MB, Richardson RB, Schoggins JW (2018) LY6E mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step. Nat Commun 9(1):1–14. https://doi.org/10.1038/s41467-018-06000-y
Merkel SF, Andrews AM, Lutton EM, Mu D, Hudry E, Hyman BT, Maguire CA, Ramirez SH (2017) Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells. J Neurochem 140(2):216–230. https://doi.org/10.1111/jnc.13861
Muntané G, Santpere G, Verendeev A, Seeley WW, Jacobs B, Hopkins WD, Navarro A, Sherwood CC (2017) Interhemispheric gene expression differences in the cerebral cortex of humans and macaque monkeys. Brain Struct Funct 222(7):3241–3254. https://doi.org/10.1007/s00429-017-1401-7
Nathwani AC, Reiss UM, Tuddenham EGD, Rosales C, Chowdary P, McIntosh J, Della Peruta M, Lheriteau E, Patel N, Raj D, Riddell A, Pie J, Rangarajan S, Bevan D, Recht M, Shen Y-M, Halka KG, Basner-Tschakarjan E, Mingozzi F, High KA, Allay J, Kay MA, Ng CYC, Zhou J, Cancio M, Morton CL, Gray JT, Srivastava D, Nienhuis AW, Davidoff AM (2014) Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 371(21):1994–2004. https://doi.org/10.1056/NEJMoa1407309
Obermeier B, Daneman R, Ransohoff RM (2013) Development, maintenance and disruption of the blood-brain barrier. Nat Med 19(12):1584–1596. https://doi.org/10.1038/nm.3407
Pardridge WM (2005) The blood-brain barrier: bottleneck in brain drug development. NeuroRx 2(1):3–14. https://doi.org/10.1602/neurorx.2.1.3
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612. https://doi.org/10.1002/jcc.20084
Pulicherla N, Shen S, Yadav S, Debbink K, Govindasamy L, Agbandje-McKenna M, Asokan A (2011) Engineering liver-detargeted AAV9 vectors for cardiac and musculoskeletal gene transfer. Mol Ther 19(6):1070–1078. https://doi.org/10.1038/mt.2011.22
Rose JA, Hoggan MD, Shatkin AJ (1966) Nucleic acid from an adeno-associated virus: chemical and physical studies. Proc Natl Acad Sci U S A 56(1):86–92
Russell RB, Alber F, Aloy P, Davis FP, Korkin D, Pichaud M, Topf M, Sali A (2004) A structural perspective on protein-protein interactions. Curr Opin Struct Biol 14(3):313–324. https://doi.org/10.1016/j.sbi.2004.04.006
Samulski RJ, Muzyczka N (2014) AAV-mediated gene therapy for research and therapeutic purposes. Ann Rev Virol 1(1):427–451. https://doi.org/10.1146/annurev-virology-031413-085355
Sweeney MD, Zhao Z, Montagne A, Nelson AR, Zlokovic BV (2019) Blood-brain barrier: from physiology to disease and back. Physiol Rev 99(1):21–78. https://doi.org/10.1152/physrev.00050.2017
Takahashi Y, Nishimura T, Higuchi K, Noguchi S, Tega Y, Kurosawa T, Deguchi Y, Tomi M (2018) Transport of pregabalin via L-type amino acid transporter 1 (SLC7A5) in human brain capillary endothelial cell line. Pharm Res 35(12):246. https://doi.org/10.1007/s11095-018-2532-0
Venkatakrishnan B, Yarbrough J, Domsic J, Bennett A, Bothner B, Kozyreva OG, Samulski RJ, Muzyczka N, McKenna R, Agbandje-McKenna M (2013) Structure and dynamics of adeno-associated virus serotype 1 VP1-unique N-terminal domain and its role in capsid trafficking. J Virol 87(9):4974–4984. https://doi.org/10.1128/JVI.02524-12
Vinogradova O, Qin J (2012) NMR as a unique tool in assessment and complex determination of weak protein–protein interactions. Top Curr Chem 326:35–45. https://doi.org/10.1007/128_2011_216
Weksler BB, Subileau EA, Perrière N, Charneau P, Holloway K, Leveque M, Tricoire-Leignel H, Nicotra A, Bourdoulous S, Turowski P, Male DK, Roux F, Greenwood J, Romero IA, Couraud PO (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19(13):1872–1874. https://doi.org/10.1096/fj.04-3458fje
Yokoyama M, Naganawa S, Yoshimura K, Matsushita S, Sato H (2012) Structural dynamics of HIV-1 envelope Gp120 outer domain with V3 loop. PLoS One 7(5):e37530. https://doi.org/10.1371/journal.pone.0037530
Yu J, Liang C, Liu S-L (2017) Interferon-inducible LY6E protein promotes HIV-1 infection. J Biol Chem 292(11):4674–4685. https://doi.org/10.1074/jbc.M116.755819
Zhang Y, Sloan SA, Clarke LE, Caneda C, Plaza CA, Blumenthal PD, Vogel H, Steinberg GK, Edwards MSB, Li G, Duncan JA, Cheshier SH, Shuer LM, Chang EF, Grant GA, Hayden Gephart MG, Barres BA (2016) Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse. Neuron 89(1):37–53. https://doi.org/10.1016/j.neuron.2015.11.013
Acknowledgments
The BBBomics database was an essential resource for transcriptional data collection used in this study (Kalari et al. 2016). The Phyre2 web portal for protein modeling, prediction, and analysis was used for protein structure prediction (Kelley et al. 2015). CABS-dock was used for molecular docking simulations (Kurcinski et al. 2015). CABS-dock was developed by the Biological and Chemical Research Center at the University of Warsaw, Poland (supported by the Foundation for Polish Science TEAM project (TEAM/2011-7/6)), co-financed by the EU European Regional Development Fund operated within the Innovative Economy Operational Program, and Polish Ministry of Science and Higher Education (IP2015 016573)). Molecular graphics and analyses were performed with the UCSF Chimera package (Pettersen et al. 2004). Chimera was developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311).
Author information
Authors and Affiliations
Contributions
Alexander Ille: Conceptualization, methodology, investigation, data curation, formal analysis, visualization, project administration, writing—original draft preparation, and writing—review and editing.
Eric Kishel: Conceptualization, methodology, formal analysis, and writing—original draft preparation.
Raoul Bodea: Investigation.
Anetta Ille: Investigation.
Hannah Lamont: Formal analysis and writing—original draft preparation.
Stacy Amico-Ruvio: Supervision, writing—original draft preparation, and writing—review and editing.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Alexander M. Ille and Eric Kishel are co-first authors.
Electronic supplementary material
Fig S1
Comparison of NVU transcriptional expression of human LY6 proteins among different age groups. (a) RMA normalized expression levels of LY6 protein transcripts in human brain NVUs from individuals age 20 to 39. The difference in expression between LY6E, LY6G5B, and LY6H is not statistically significant (n.s.); n = 3. (b) RMA normalized expression levels of LY6 protein transcripts in human brain NVUs from individuals age 40 to 59; n = 5. (c) RMA normalized expression levels of LY6 protein transcripts in human brain NVUs from individuals age 60 to 79. The difference in expression between LY6E and LY6H is not statistically significant (n.s.); n = 4. Data shown as means ± SEM; *p < 0.05; ****p < 0.0001; 1-way ANOVA (PNG 342 kb)
Rights and permissions
About this article
Cite this article
Ille, A.M., Kishel, E., Bodea, R. et al. Protein LY6E as a candidate for mediating transport of adeno-associated virus across the human blood-brain barrier. J. Neurovirol. 26, 769–778 (2020). https://doi.org/10.1007/s13365-020-00890-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13365-020-00890-9