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Simultaneous CAR- and alpha V integrin-binding ablation fails to reduce Ad5 liver tropism
Karine Martin1, Anne Brie, Patrick Saulnier
1UMR1582 CNRS/IGR/Aventis-Gencell, Villejuif Cedex, France.
Abstract:
Targeting adenovirus encoding therapeutic genes to specific cell types has become a major goal in gene therapy. Coxsackievirus and adenovirus receptor (CAR) and alpha(V) integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. Redirecting Ad5-based vectors requires abrogation of the natural interaction between the viral capsid and its cellular receptors and simultaneous introduction of a new binding specificity into the viral capsid. To abrogate native Ad5 tropism, fiber knob mutations Pro409Glu and Lys417Ala were each incorporated into adenoviral vectors, while the RGD motif was deleted from the penton base. In vitro transduction experiments showed that these capsid mutations eliminated Ad5 interactions with CAR and alpha(V) integrins. Moreover, incorporation in the fiber HI loop of a vitronectin-derived ligand (VN4) specific for the uPAR/CD87 receptor provided the Lys417Ala virus with an alternative entry pathway specific for uPAR-expressing cells, indicating a successful in vitro retargeting of the vector. Unexpectedly, however, simultaneous disruption of Ad5 binding to CAR and alpha(V) integrins had no effect on liver gene transfer following systemic administration in mice. This study highlights the need to understand better the molecular determinants involved in adenovirus uptake by the liver to control the fate of adenoviral vectors in vivo.
Insights
Researchers modified adenovirus vectors to target specific cells for gene therapy. While successful in vitro, disrupting natural Ad5 interactions did not alter liver gene transfer in vivo, highlighting unknown liver uptake mechanisms.
Area of Science:
- Gene Therapy
- Virology
- Molecular Biology
Background:
- Adenovirus type 5 (Ad5) vectors are crucial for gene therapy, but their natural tropism limits targeted delivery.
- Ad5 interacts with Coxsackievirus and adenovirus receptor (CAR) and alpha(V) integrins for cell entry.
- Retargeting Ad5 vectors requires blocking native interactions and introducing new cell-specific binding.
Purpose of the Study:
- To investigate methods for retargeting adenovirus type 5 (Ad5) vectors.
- To abrogate native Ad5 interactions with CAR and alpha(V) integrins.
- To assess the in vivo impact of modified Ad5 vectors on liver gene transfer.
Main Methods:
- Engineered Ad5 vectors with fiber knob mutations (Pro409Glu, Lys417Ala) and penton base RGD deletion.
- Incorporated a vitronectin-derived ligand (VN4) into the fiber HI loop for uPAR/CD87 receptor targeting.
- Evaluated in vitro transduction efficiency and in vivo liver gene transfer after systemic administration in mice.
Main Results:
- Capsid mutations successfully eliminated Ad5 binding to CAR and alpha(V) integrins in vitro.
- Retargeted vector (Lys417Ala with VN4) demonstrated specific uPAR-expressing cell transduction in vitro.
- Disrupting Ad5 binding to CAR and alpha(V) integrins did not affect liver gene transfer in vivo.
Conclusions:
- Adenovirus vector retargeting is feasible in vitro by modifying capsid-receptor interactions.
- The study reveals that CAR and alpha(V) integrins are not the primary determinants of Ad5 liver tropism in vivo.
- Further research is needed to elucidate the molecular mechanisms governing adenovirus uptake by the liver.