Pete Ward

Tumor-Specific, Replication-Competent Adenovirus Vectors Overexpressing the Adenovirus Death Protein

ABSTRACT We have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting the adp gene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutant dl1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G0 to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus, dl1101/1107, or wild-type Ad. They are also more efficient than dl1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1− E3−replication-defective vector expressing β-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10−4 PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected with dl1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.

Novel cis-Acting Replication Element in the Adeno-Associated Virus Type 2 Genome Is Involved in Amplification of Integrated rep-cap Sequences

ABSTRACT This study identifies a region of the adeno-associated virus type 2 (AAV-2) rep gene (nucleotides 190 to 540 of wild-type AAV-2) as a cis-acting Rep-dependent element able to promote the replication of transiently transfected plasmids. This viral element is also shown to be involved in the amplification of integrated sequences in the presence of adenovirus and Rep proteins.

Rep-Dependent Initiation of Adeno-Associated Virus Type 2 DNA Replication by a Herpes Simplex Virus Type 1 Replication Complex in a Reconstituted System

ABSTRACT Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.

Characterization of the Mouse Adeno-Associated Virus AAVS1 Ortholog

ABSTRACT The nonpathogenic human adeno-associated virus (AAV) has developed a mechanism to integrate its genome into human chromosome 19 at 19q13.4 (termed AAVS1), thereby establishing latency. Here, we provide evidence that the chromosomal signals required for site-specific integration are conserved in the mouse genome proximal to the recently identified Mbs85 gene. These sequence motifs can be specifically nicked by the viral Rep protein required for the initiation of site-specific AAV DNA integration. Furthermore, these signals can serve as a minimal origin for Rep-dependent DNA replication. In addition, we isolated the mouse Mbs85 proximal promoter and show transcriptional activity in three mouse cell lines.

Rescue of the Adeno-Associated Virus Genome from a Plasmid Vector: Evidence for Rescue by Replication

ABSTRACT In cultured cells, adeno-associated virus (AAV) replication requires coinfection with a helper virus, either adenovirus or herpesvirus. In the absence of helper virus coinfection AAV can integrate its genome site specifically into the AAVS1 region of chromosome 19. Upon subsequent infection with a helper virus, the AAV genome is released from chromosome 19 by a process termed rescue, and productive replication ensues. The AAV genome cloned into a plasmid vector can also serve to initiate productive AAV replication. When such constructs are transfected into cells and those cells are simultaneously or subsequently infected with a helper virus, the AAV genome is released from the plasmid. This process is thought to serve as a model for rescue from the human genomic site. In this report we present a model for rescue of AAV genomes by replication. A hallmark of this model is the production of a partially single-stranded and partially double-stranded molecule. We show that the AAV2 Rep 68 protein, together with the UL30/UL42 herpes simplex virus type 1 DNA polymerase and the UL29 single-strand DNA binding protein ICP8, is sufficient to efficiently and precisely rescue AAV from a plasmid in a way that is dependent on the AAV inverted terminal repeat sequence.

From Obligation to Consumption in Two-and-a-half Hours: A Visual Exploration of the Sacred with Young Polish Migrants

This article reports the findings of a visual ethnographic case study that explored the shifting nature of religious belief in contemporary Britain due to the migration of young Polish people. The focus on studying what is ‘sacred’ to Polish migrants revealed how their religious beliefs and practices are changing in a new religious context. Surprised by the plurality of beliefs and the side-lined church, Polish migrants find that they are no longer obligated to practise their religion and must come to terms with choosing how to be religious. Locating the sacred in church, family, and nature, they search for a sense of home and stability within their new social context. Traditional beliefs in God, Jesus, Mary, and angels still form the basis for personal meaning making, but the practice of religion in the context of the UK is often altered in response to increased options for religious practice, including not practising at all.

Blueprint ecclesiology and the lived : normativity as perilous faithfulness.

Normativity in Ecclesiology has tended to be based on a particular understanding of theology as blueprint. In the Ecclesiology and Ethnography Conversation there has been some dispute around how theological normativity should operate. This paper argues that theological knowledge arises from an ecclesial context of ‘abiding.’ This abiding is pneumatological in nature ‘like the wind’ and as such it is perilous. This point is argued with resort to a critical realist epistemology.

Practical Theology and the Ordinary

Abstract This article examines the idea of ordinary theology using visual ethnographic methodologies. It is the third in a series of papers arising from research into Migration and Visual Culture among Polish Catholic young people. Using the work of Raymond Williams as a starting point the theme of the ordinary in practical theology is examined. The article argues for the ordinary as being constructed in relation to formal theology as well as part of a whole way of life.

Liquid ecclesiology : the Gospel and the Church.

In Liquid Ecclesiology Pete Ward explores the theological contours of the turn to ethnography in the study of the Christian Church. His approach rests on a theology of culture that holds in tension and paradox the expression of the Church and divine presence. This theological framework is then developed through an extended qualitative empirical case study examining the communicative practices of the contemporary evangelical Church. The case study examines how the evangelical Gospel through expression has become marginalised in the everyday life of communities being replaced by a new more individual and personalised theology seen in worship songs. The final section of the book returns to the debates around ethnographic forms of theology and the question of normativity. This book will be of interest to all those engaged in empirical and theological work, as well as those researching the contemporary Church and evangelicalism

Ordinary Theology as Narratives: An Empirical Study of Young People's Charismatic Worship in Scotland

Abstract Through a detailed analysis this paper demonstrates the usefulness of investigating public worship events as a site for studying the construction of ordinary theology. By identifying the theological narratives being enacting and affirmed by a specific group in such contexts helps strengthen our understanding of the documentary of a specific community. It also illustrates how the particular of a given culture is linked to and used to situate the larger narrative tradition. We suggest more studies of narrative and flow of worship events would benefit Practical Theology to help reveal new aspects of construction and flow of ordinary theology for evangelical and charismatics groups. Zusammenfassung Mit Hilfe einer detaillierten Analyse veranschaulicht dieser Beitrag den Wert der Erforschung öffentlicher Gottesdienste fü die Auseinandersetzung mit der Entstehung und Vermittlung von Laien-Theologie. Durch die Rekonstruktion der theologischen Erzählungen, die durch die Handlungen der an solchen Ereignissen Beteiligten erzeugt und bestätigt werden, gelingt es, das theologische Verständnis einer spezifischen Gemeinschaf und seiner Formen der Darstellung besser zu verstehen. Außerdem wird so illustriert, wie das Spezifische einer gegebenen Kultur mit allgemeineren narrativen Traditionen verbunden ist und auf diese bezogen wird. Es wird deshalb vorgeschlagen, weitere Studien über die Erzählungen und den Ablauf von Gottesdiensten durchzuführen, damit die Praktische Theologie gewinnbringend neue Aspekte der Konstruktion und Gestaltung der Laien-Theologie im Bereich evangelikaler und charismatischer Gruppierungen zu entdecken vermag.