Rational and random approaches to adenoviral vector engineering (2011)

The overall aim of this thesis is to contribute to the engineering of more selective and effective oncolytic Adenovirus (Ad) vectors. Two general approaches are taken for this purpose: (i) genetic capsid modification to achieve Ad retargeting (Chapters 2 to 4), and (ii) directed evolution to improve the cytolytic potency of Ad (Chapter 5). In order to provide some context for these approaches, Chapter 1, part II gives a brief background on Ad biology and vectorology. Further, in Chapter 1, part III, a broad overview is provided of the ways that evolution-based engineering has previously been used to generate or improve viral vectors.\n\nChapters 2 and 3 focus on the modification of the minor Ad capsid protein IX (pIX). pIX is present on the faces of the Ad capsid icosahedron, functioning as ‘cement’ between the much larger hexon proteins. Previously, the C-terminus of pIX proved serviceable as an anchor for the genetic capsid incorporation of targeting ligands and other heterologous moieties. In Chapter 2, a new system is described that allows for the rapid functional testing of new pIX-ligand fusion proteins. In this system, lentiviral vectors are used to generate cells stably expressing the pIX variant of interest. Large-scale infection on such cells with a pIX-deleted Ad vector subsequently yields an Ad vector preparation phenotypically pseudotyped with the new pIX variant. This system thus allows rapid analysis of new pIX-ligand fusions in the context of the Ad capsid without having to genetically modify the Ad genome. In Chapter 3, the lentiviral vector-based pIX-pseudotyping system is put to use for the analysis of a new pIX fusion protein harboring a single-chain T-cell receptor (scTCR) as a targeting ligand. The concerning scTCR was directed against the intracellular cancer-testis antigen melanoma-associated antigen-A1. Importantly, this chimeric pIX molecule proved to be efficiently incorporated into the Ad capsid. Moreover, Ad transduction studies showed evidence of the capsid-displayed scTCR to mediate a degree of specific target cell transduction via the cognate peptide-MHC complex. \n\nAnalogously as done for pIX, Chapter 4 describes a phenotypical pseudotyping approach for fiber. The Ad-encoded fiber protein is present as a trimeric rod-like structure that extends from the vertices of the Ad capsid icosahedron. Its outward-facing, C-terminal ‘knob’ domain is responsible for binding the Coxsackie and adenovirus receptor (CAR), Ad’s in vitro primary cell surface attachment protein. With its prominent role in native receptor binding, the Ad fiber is logically subject to many capsid modification strategies that aim at altering Ad tropism. Thus to facilitate expedited testing of new fiber variants, a lentiviral vector-based, fiber-pseudotyping system was set up. This involved optimization of the fiber (variant) expression cassettes by inclusion of the tripartite leader sequence of Ad’s major late transcription unit. A second objective of this study was to functionally assess a new chimeric fiber harboring a tumor antigen-directed single-chain variable fragment (scFv) antibody. Although this fiber variant showed some degree of target binding and formed stable trimers, it displayed problems regarding capsid incorporation ability, functionality within the capsid, and folding of its scFv constituent. Thus, this particular fiber proved not suitable for Ad retargeting. \n\nFinally, Chapter 5 describes the development and validation of a novel evolution-based engineering approach for Ad. To date, most Ad-based vectors have been generated through molecular design. Although this rational tailoring of Ad has led to significant vector improvements, it is often still hampered by our limited understanding of the intricate viral function-structure relationships. Therefore, ‘random’ virus engineering strategies (see Chapter 1, part III) may be a useful alternative or complementary approach for the generation of new or improved viral vectors. In this regard, the high mutation rates of RNA viruses have proven readily exploitable in adaptation studies to achieve vectorological goals. Thus, it was hypothesized that a mutator Ad polymerase-based, ‘accelerated evolution’ procedure would likewise be of use for Ad vector engineering. To develop such a system, the intrinsic mutation rate of Ad replication was sought to be increased by modification of the Ad-encoded DNA polymerase (Ad pol). This was done by mutation of residues within regions putatively important for nucleotide selection or proofreading. A mutation-accumulation and deep sequencing strategy was subsequently used to identify any mutators among the Ad pol mutants. Finally, the mutator polymerase-based directed evolution approach was validated by conducting an evolution procedure aimed at increasing Ad’s oncolytic potency, and by subsequent characterization of resultant bioselected virus populations and isolated clones.