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A system for the propagation of adenoviral vectors with genetically modified receptor specificities

Nature Biotechnology volume 17, pages 470–475 (1999)Cite this article

Abstract

The development of genetically modified adenovirus (Ad) vectors with specificity for a single cell type will require both the introduction of novel tropism determinants and the ablation of endogenous tropism. Consequently, it will not be possible to exploit the native cellular entry pathway in the propagation of these targeted Ad vectors. Based on the concept that Ad enters cells by a two-step process in which a primary receptor serves as a high affinity binding site for the Ad fiber knob, with subsequent internalization mediated by αv integrins, we designed two artificial primary receptors. The extracellular domain of one of these synthetic receptors was derived from a single-chain antibody (sFv) with specificity for Ad5 knob, while the second receptor consisted of an icosapeptide identified by biopanning a phage display library against Ad5 knob. Expression of either of these artificial virus-binding receptors in fiber receptor-negative cells possessing αv integrins conferred susceptibility to Ad infection. We then created a novel mechanism for cell binding by genetically modifying both the vector and the target cell. In this approach, six histidine (His) residues were incorporated at the C-terminal of the Ad fiber protein. The resultant Ad vector was able to infect nonpermissive cells displaying the cognate artificial receptor, containing an anti-His sFv. This strategy, comprising a genetically engineered Ad virion and a modified cell line, should be useful in the propagation of targeted Ad vectors that lack the ability to bind the native fiber receptor.

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Figure 1: (A) Rationale for an artificial receptor to provide a fiber-receptor independent pathway of cellular entry for Ad vectors lacking native tropism.
Figure 2: Ad5KsFv.rec functions as an artificial receptor for fiber receptor-independent Ad5 infection of HeLa cells. Cells were mock-transfected or transfected with pAd5KsFv.rec or pHook, which expresses an irrelevant receptor, designated phOxsFv.rec. Forty-eight hours posttransfection, the cells were preincubated with PBS or with Ad2 or Ad5 knob prior to infection with AdCMV.
Figure 3: Ad5KsFv.rec functions as an artificial receptor for Ad5 infection of nonpermissive U118MG cells.
Figure 4: MH20.rec functions as an artificial receptor for Ad5 infection of nonpermissive NR6 cells.
Figure 5: A specific interaction between the C-terminal His tag of the fiber protein of a modified virus and an artificial His tag receptor mediates infection of nonpermissive U118MG cells.
Figure 6: A specific interaction between the C-terminal His tag of the fiber protein and a His tag receptor permits propagation of Ad in U118MG cells.

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Acknowledgements

We thank Dr Andreas Plückthun (University of Zurich, Zurich, Switzerland) for the generous gift of pAK100His2 and Dr Stefan Dübel (German Cancer Research Center, Heidelberg, Germany) for helpful discussions and advice concerning the expression and purification of the anti-Ad5 knob sFv. This research was supported by grants from the Muscular Dystrophy Association to J.T.D., the American Heart Association to D.T.C. and J.T.D., and from the American Lung Association and the National Institutes of Health (R01 CA 74242 and R01 HL 50255) to D.T.C.

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Authors and Affiliations

  1. Gene Therapy Center,The University of Alabama at Birmingham , Birmingham, 35294, AL

    C. Ryan Miller, Myunghee Kim, Igor Dmitriev & Galina Mikheeva

  2. Department of Medicine, Division of Pulmonary and Critical Care Medicine, The University of Alabama at Birmingham, Birmingham, 35294, AL

    Joanne T. Douglas & David T. Curiel

  3. Department of Radiation Oncology, The University of Alabama at Birmingham, Birmingham, 35294 , AL

    Victor Krasnykh

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Correspondence to David T. Curiel.

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Douglas, J., Miller, C., Kim, M. et al. A system for the propagation of adenoviral vectors with genetically modified receptor specificities. Nat Biotechnol 17, 470–475 (1999). https://doi.org/10.1038/8647

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