In the realm of gene therapy, the efficient delivery of genes to specific cells in vivo remains a significant challenge. Among the arsenal of vectors available for gene therapy, adenovirus (Ad) stands out for its safety profile, well-established characteristics, and ease of production. However, Ad’s broad tropism, primarily relying on the coxsackie B virus and Ad receptor (CAR) and major histocompatibility complex class I alpha-2 domain, poses limitations as it fails to transduce cells lacking these receptors.
The cellular attachment of Ad is facilitated by the fiber protein, a homotrimer crucial for binding to both the viral capsid and host cell receptors. Altering Ad’s tropism, a process known as viral retargeting, can be achieved through two main strategies: nongenetic and genetic retargeting. Genetic retargeting involves modifying viral proteins, with efforts including chimeric fibers, penton base modifications, and new ligand insertions. However, genetic alterations can impact the trimeric structure of the fiber, limiting the extent of modifications that can be made without compromising functionality.
In a groundbreaking study, a novel approach to genetically retarget Ad was introduced, termed “deknobbing,” which involves removing the fiber sequence beyond the seventh shaft repeat. This innovative strategy eliminates the cell-binding ligand while disrupting the trimerization signal. To address the trimerization loss, an external trimerization motif, the neck region peptide (NRP) of human lung surfactant protein D, was inserted. Additionally, a new cell-binding ligand, the arginine-glycine-aspartic acid (RGD) motif targeting integrins αvβ3 and αvβ5, was incorporated. The resulting recombinant fibers demonstrated successful trimerization, nuclear localization, and penton formation, highlighting their potential for retargeting Ad to cells expressing integrins but lacking CAR.
Characterizing the functionality of these novel fibers involved assessing their trimerization, glycosylation, and assembly with penton base to form complete pentons. Despite variations in shaft lengths among different recombinant fibers, all demonstrated trimer formation, assembly with penton base, and nuclear localization. Interestingly, the glycosylation status revealed that O-GlcNAc residues, present in the WT fiber, were dispensable for essential fiber functions. The efficient assembly of penton capsomers with the knobless fibers suggested enhanced stability compared to WT fibers, particularly in insect cells.
Further analysis of the recombinant fibers’ binding properties showcased their specificity for integrins αvβ3 and αvβ5, with the RGD motif serving as a key mediator of this interaction. Competition assays with WT penton base confirmed the RGD-specific binding of the recombinant fibers to integrins. These findings underscored the successful genetic retargeting of Ad using deknobbing and external trimerization motifs, paving the way for enhanced viral tropism and cell specificity in gene therapy applications.
The production and purification of these innovative fibers involved cloning into vectors, expression in cell lines, and chromatographic purification steps. The rigorous phenotypic characterization encompassing cellular localization, trimerization, assembly with penton base, and binding specificity provided comprehensive insights into the functional attributes of the recombinant fibers. Notably, the R7-RGD fiber emerged as the most promising candidate for reintroduction into the viral genome, highlighting its potential for generating knobless Ad vectors with enhanced targeting capabilities.
In conclusion, the strategic genetic modification of Adenovirus fiber through deknobbing represents a significant advancement in the field of gene therapy. By harnessing external trimerization motifs and novel cell-binding ligands, researchers have successfully engineered fibers with expanded tropism and enhanced binding specificity. These findings hold immense promise for the development of next-generation Ad vectors tailored for precise gene delivery to target cells, overcoming the limitations of broad tropism associated with conventional Ad vectors.
Key Takeaways
– Genetic retargeting through deknobbing of Ad fiber offers a novel strategy for enhancing viral tropism in gene therapy applications.
– External trimerization motifs and cell-binding ligands enable the construction of knobless Ad vectors with improved targeting capabilities.
– Phenotypic characterization demonstrates the successful trimerization, assembly, and binding specificity of recombinant fibers, supporting their potential for enhanced gene delivery.
– The R7-RGD fiber emerges as a promising candidate for reintroduction into the viral genome, paving the way for the development of precision-targeted Ad vectors.
Tags: chromatography, upstream, gene therapy, regulatory, downstream, biotech, monoclonal antibodies
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