Directing adenovirus across the blood–brain barrier via melanotransferrin (P97) transcytosis pathway in an in vitro model

Abstract

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 intracranial 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 redirect 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.

Stem and progenitor cell-mediated tumor selective gene therapy

“…The presumption that must be made with these types of targeted approaches is that proteins (or other surface molecules) can be identified, which are sufficiently unique in level or pattern of expression to serve as 'specific receptors' for the targeting moiety. In antitumor approaches, for example, numerous constructs have been reported that target transferrin receptor, [39][40][41][42][43][44][45] since this receptor has been reported to be expressed at levels approximately twofold higher in tumors than in normal tissues. 46,47 Intuitively, a twofold difference may not provide a sufficient differential between tumor and normal tissue to confer tumor selectivity, unless the dose-response curve of the therapeutic entity is very steep.…”

Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication‐competent CD95L adenovirus

“…Thus the lack of E1B and mutated E1A may further restrict propagation of Ad5-FFE-02. Melanocytes of the substantia nigra are also protected from adenoviral infection by the blood ⁄ brain barrier (53).…”

Agile delivery of protein therapeutics to CNS

“…Indeed, Tang and colleagues engineered a fusion protein of sMTf and cell surface coxsackie-adenovirus receptor (sCAR), which binds as adaptor protein to both adenovirus serotype 5 (Ad5) and brain endothelium receptor and promotes internalization of the Ad5 vector. Using an in vitro BBB model, these authors demonstrated that sMTf-sCAR fusion protein redirects the cell trafficking of the Ad5 vector and facilitates its transport across the brain endothelial cells [294]. Moreover, sMTf was able to successfully deliver conjugated doxorubicin molecule to brain tumors in vivo [295].…”