Nonhuman primate adenoviruses have formed a valuable alternative for the use of human adenoviruses in vaccine development and gene therapy strategies by virtue of the low seroprevalence of... Show moreNonhuman primate adenoviruses have formed a valuable alternative for the use of human adenoviruses in vaccine development and gene therapy strategies by virtue of the low seroprevalence of neutralizing immunity in the human population. The more recent use of several human adenoviruses as oncolytic agents has exhibited excellent safety profiles and firm evidence of clinical efficacy. This raises the question whether nonhuman primate adenoviruses could also be employed for viral oncolysis in human patients. The research in this thesis provides a rational and data-supported decision on the use of nonhuman primate adenoviruses as a base for the development of new oncolytic derivatives with limited neutralizing immunity in the human population. Moreover, the development of a potent new gorilla-derived oncolytic adenovirus named GoraVir shows the feasibility of the approach. Hopefully, this research provides some reassurance regarding the future use of replication-competent nonhuman primate adenovirus vectors as therapeutic agents. Show less
Oncolytic viruses are highly promising agents for cancer therapy. Reovirus type 3 Dearing and adenovirus type 5 are potent representatives of this new type of treatment. They have been tested... Show moreOncolytic viruses are highly promising agents for cancer therapy. Reovirus type 3 Dearing and adenovirus type 5 are potent representatives of this new type of treatment. They have been tested in a multitude of clinical trials. In these studies reovirus and adenovirus display a good safety profile, but anti-tumour efficacy should be improved. The work described in this thesis mainly concentrated on improving the entry and spread of reoviruses and adenoviruses in tumours by means of genetic modification of the viruses. Show less
In this thesis, new insights in the complex and intertwined relationship between viral infections, T cells and natural killer cells after allogeneic HSCT in children are provided and discussed.... Show moreIn this thesis, new insights in the complex and intertwined relationship between viral infections, T cells and natural killer cells after allogeneic HSCT in children are provided and discussed. Patients are at high risk of viral complication during the T cell deficient period early after HSCT. When viral infections occur, interventions to bridge the period until the recovery of antiviral T cell immunity are of great importance to improve the clinical outcome after HSCT. Besides antiviral medication and the use of adoptive immunotherapy, NK cells may play a role in the protection against viruses when T cells are not available to perform their task. Show less
Eby, J.M.; Barse, L.; Henning, S.W.; Rabelink, M.J.W.E.; Klarquist, J.; Gilbert, E.R.; ... ; Poole, I.C. le 2017
Accurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA... Show moreAccurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA template into target cells. The exogenous template, called donor DNA, normally contains the foreign sequences flanked by DNA regions sharing sequence identity ("homologous") to those bracketing the target site. The strategies for mediating the formation of DSBs at the predefined genomic loci, have been undergoing intense investigation since the introduction of sequence-customizable zinc-finger nuclease (ZFN) technology. More recently, prokaryotic protein-based transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases (RGNs) derived from CRISPR-associated protein (Cas9) complexes have substantially broadened the availability and applicability of designer nuclease-mediated genome editing. A potential alternative research line to the use of designer nucleases, is to investigate whether specific DNA structures can, by themselves, serve as triggers of the DNA damage response and, in doing so, elicit targeted gene repair. Such an approach would simplify genome editing protocols, such as, by reducing the number of reagents needed to be introduced into target cells. In this thesis, the roles of these secondary structures as well as designer nucleases and donor-DNA templates, delivered via adenoviral vectors, is described. Show less
Osteosarcoma and Ewing sarcoma are the most common bone cancers in children and young adults. Despite advanced surgical techniques and multi-drug chemotherapy, patients with recurrent, metastatic... Show moreOsteosarcoma and Ewing sarcoma are the most common bone cancers in children and young adults. Despite advanced surgical techniques and multi-drug chemotherapy, patients with recurrent, metastatic or chemotherapy-resistant disease have a poor outcome. Thus, novel targeted therapies are needed that combine potent and specific anti-cancer activity with limited toxicity toward normal tissues. The thesis is introduced by an outline of the biological properties of osteosarcoma and Ewing sarcoma, followed by an overview of cancer immunology and immunotherapy with the primary focus on innate immunity of human natural killer (NK) cells and macrophages. In the research chapters, cellular interactions of NK cells and macrophages with bone tumor cells are characterized in order to achieve favorable effects on anti-cancer immune cell functions. It is demonstrated that the anti-cancer potential of especially NK cells but also macrophages can be enhanced and directed to the bone tumor cells. It is discussed that the modulation of tumor__immune cell interactions may help to design novel immunotherapeutic approaches to harness anti-cancer functions of innate immune cells against osteosarcoma and Ewing sarcoma. Show less
Recombinant viral vectors hold great promise in the field of cancer gene therapy. While a plethora of viruses is being evaluated as oncolytic agents, human adenoviruses of serotype 5 (HAdV-5) are... Show moreRecombinant viral vectors hold great promise in the field of cancer gene therapy. While a plethora of viruses is being evaluated as oncolytic agents, human adenoviruses of serotype 5 (HAdV-5) are among the most popular of viruses to be developed. Although clinical studies have demonstrated safety of cancer gene therapy with HAdV-5-derived vectors, the efficacy still needs further enhancement. Several factors have been identified that limit the anti-tumor efficacy. One major bottleneck is the inadequate penetration and spread of the virus within the tumor. This is attributable, at least in part, to the low or heterogeneous expression of the coxsackie and adenovirus receptor (CAR) on the tumor cells. This thesis describes the development and preclinical evaluation of novel tumor-targeted HAdV-5 vectors, through implementing the genetic fusion of capsid proteins (protein IX and fiber) with a variety of tumor-targetin g polypeptides. Show less
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... Show moreThe 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. Chapters 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. Analogously 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. Finally, 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. Show less
In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the... Show moreIn this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the fact that many for gene therapy interesting cell types do not, or only at low levels express the CAR receptor, necessary for infection. We developed a linker protein consisting of the virus-binding moiety of CAR genetically fused to the chicken protein avidin. Biotinylated ligands for cell specific receptors are bound to the linker protein via the avidin-biotin interaction. This now targeting protein is used to redirect adenovirus vectors to previously refractory cell types. Using this technology endothelial cell lines as well as primary endothelial cells can by infected at low MOI__s using an biotinylated cyclic RGD peptide. Primary bone marrow derived macrophages and macrophage cell lines are easily infected using a biotinylated dA6dG10 oligo nucleotide ligand. In vivo experiments showed a marked reduction of adenovirus mediated transgene expression by the liver, the organ responsible for virus uptake when unmodified adenovirus vectors are administered, after addition of several different ligands to the virus via the linker protein. Show less
Real-time monitoring of PCR has strongly supported the increased diagnostic use of nucleic acid detection assays in clinical virology. Particularly the improvements in the ability to quantify... Show moreReal-time monitoring of PCR has strongly supported the increased diagnostic use of nucleic acid detection assays in clinical virology. Particularly the improvements in the ability to quantify target nucleic acid sequences offer new opportunities in the management of viral infections. Real-time PCR is rapidly replacing traditional PCR, and new diagnostic uses will likely emerge. This thesis explores the wide range of potential applications of real-time quantitative PCR technology in clinical virology. This exploration is directed to the design of methods, the application to relevant patient categories, the comparison with established methods where available, and the definition of the clinical relevance of the approach. The focus comprises viral targets where an elaborate balance between viral replication and the host immune system has been established, which brings about viral maintenance without affecting the host, until this balance is disturbed. Show less
Current generation adenoviral vectors (Ads) are not suitable for those gene therapy approaches that require long-term gene expression. This is due to their high immunogenicity and transient gene... Show moreCurrent generation adenoviral vectors (Ads) are not suitable for those gene therapy approaches that require long-term gene expression. This is due to their high immunogenicity and transient gene expression in fast dividing tissue. The development of gutless Ads, also known as helper-dependent Ads, is a major improvement in reducing the immunogenicity of the vector system. Gutless Ads lack virtually all viral protein-coding sequences, thus severely limiting the viral-antigen evoked cellular immune responses that may result in the elimination of the transduced cells. Safety wise, recombinant Ads are considered safe due to their inability to replicate autonomously. However, we show in this thesis that replication of recombinant Ads can be rescued by the co-infection of wild type (wt) Ads. In this thesis studies are described that aim at the development of a new system to prevent vector mobilization. Though at its present state not directly applicable, this system could also potentially be used for the production of gutless Ads devoid of helper Ad contamination. To improve efficacy of the Ad vector in dividing tissue we also studied two integration systems for their applicability in Ads. Overall, the experiments described in this thesis aim at generating safer vectors that should result in prolonged transgene expression due to lower immunogenicity and genomic integration. Show less
This thesis centers on the mechanisms of estrogen action and the effects on the development of atherosclerosis. We have focused on the liver as central organ in lipid and glucose metabolism and the... Show moreThis thesis centers on the mechanisms of estrogen action and the effects on the development of atherosclerosis. We have focused on the liver as central organ in lipid and glucose metabolism and the vessel wall as the actual site where the injury occurs. To gain insight in tissue-specific actions of estrogens, we have spent considerable effort to develop tools for liver and blood vessel specific modulation of the estrogen receptor (ER) signaling cascade. The generation, characterization and application of these tools in vitro and in vivo will be described in the different chapters of this thesis. Show less
The adenoviral vector is one of the most used viral vectors for gene therapy applications. The specificity towards its primary receptor is a limitation of the use of HAdV as therapeutical vector,... Show moreThe adenoviral vector is one of the most used viral vectors for gene therapy applications. The specificity towards its primary receptor is a limitation of the use of HAdV as therapeutical vector, as not all cells have these primary receptors on their surface. To overcome this problem scientists have started to change the receptor specificity of the HAdV vectors by introducing heterologous polypeptides. One of the most promising candidates to function as anchor for these heterologous polypeptides is the minor capsid protein IX (pIX). Alpha-helical spacers of different lengths have been inserted between pIX and a polypeptide ligand. The results showed that an increase in spacer length positively correlated with the accessibility of the peptide on the capsid surface for antibodies and cellular membrane proteins. Next, a hyper stable scFv directed against __-galactosidase was incorporated in the virion via fusion to pIX. These data were the first to show incorporation of a hyper-stable scFv into the HAdV, and that was functional on the capsid surface. Beside the technical approach to use pIX as anchor for heterologous polypeptides here also the function of pIX in the capsid has been studied. This study has led to the model in which the capsid stability mediated by pIX might not be the result of homo-multimerization of this molecule. Show less
