Directing adenovirus across the blood-brain barrier via melanotransferrin (P97) transcytosis pathway in an in vitro model.
Adenovirus serotype 5 (Ad5) is widely used in the development of gene therapy protocols. However, current gene therapy strategies involving brain are mostly based on intra-cranial injection. A major obstacle for systemically administered vectors to infect brain tissue is the blood-brain barrier (BBB). One strategy to cross the BBB is transcytosis, a transcellular transport process that shuttles a molecule from one side of the cell to the other side. Recently, melanotransferrin (MTf)/P97 was found to be able to cross the BBB and accumulate in brain. We thus hypothesize that re-directing Ad5 vectors to the MTf transcytosis pathway may facilitate Ad5 vectors to cross the BBB. To test this hypothesis, we constructed a bi-specific adaptor protein containing the extracellular domain of the coxsackie-adenovirus receptor (CAR) and the full-length melanotransferrin (sCAR-MTf), and investigated its ability to re-direct Ad5 vectors to the MTf transcytosis pathway. We found this adaptor protein could re-direct Ad5 to the MTf transcytosis pathway in an in vitro BBB model, and the transcytosed Ad5 viral particles retained their native infectivity. The sCAR-MTf-mediated Ad5 transcytosis was temperature- and dose dependent. In addition, we examined the directionality of sCAR-MTf-mediated Ad5 transcytosis, and found the efficiency of apical-to-basal transcytosis was much higher than that of basal-to-apical direction, supporting a role of this strategy in transporting Ad5 vectors towards the brain. Taken together, our study demonstrated that re-directing Ad5 to the MTf transcytosis pathway could facilitate gene delivery across the BBB.
Tang
Y
Y
Division of Human Gene Therapy, Department of Medicine, The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Han
T
T
Everts
M
M
Zhu
Z B
ZB
Gillespie
G Y
GY
Curiel
D T
DT
Wu
H
H
D000256:Adenoviridae / Q000235:genetics*; D000257:Adenoviridae Infections / Q000378:metabolism*; D000951:Antigens, Neoplasm; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D011991:Receptors, Virus / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods
D000951:Antigens, Neoplasm; C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D008156:Luciferases