David Onion

Viral gene therapy strategies: from basic science to clinical application

A major impediment to the successful application of gene therapy for the treatment of a range of diseases is not a paucity of therapeutic genes, but the lack of an efficient non‐toxic gene delivery system. Having evolved to deliver their genes to target cells, viruses are currently the most effective means of gene delivery and can be manipulated to express therapeutic genes or to replicate specifically in certain cells. Gene therapy is being developed for a range of diseases including inherited monogenic disorders and cardiovascular disease, but it is in the treatment of cancer that this approach has been most evident, resulting in the recent licensing of a gene therapy for the routine treatment of head and neck cancer in China. A variety of virus vectors have been employed to deliver genes to cells to provide either transient (eg adenovirus, vaccinia virus) or permanent (eg retrovirus, adeno‐associated virus) transgene expression and each approach has its own advantages and disadvantages. Paramount is the safety of these virus vectors and a greater understanding of the virus–host interaction is key to optimizing the use of these vectors for routine clinical use. Recent developments in the modification of the virus coat allow more targeted approaches and herald the advent of systemic delivery of therapeutic viruses. In the context of cancer, the ability of attenuated viruses to replicate specifically in tumour cells has already yielded some impressive results in clinical trials and bodes well for the future of this approach, particularly when combined with more traditional anti‐cancer therapies. Copyright

Assays of proteasome activity in relation to aging.

Proteasomes play a major role in intracellular protein turnover. They exist in cells in several different molecular forms including 20S proteasomes, 26S proteasomes and PA28-20S proteasome complexes. In this study we have compared the properties of these purified proteasome complexes to try to design assays that will distinguish between the different complexes (26S proteasome, 20S proteasome, PA28-20S proteasome) in cell extracts. Although the different purified complexes were found to have differences in stability, and in their sensitivity to low concentrations of SDS and salt, the results suggest that it is not straightforward to assay selectively for each type of complex in cell extracts. The relative contribution of different proteasome complexes varies in different cell types and there may be other proteases present which hydrolyse the chosen substrate. Proteasome assays carried out under defined conditions allow comparisons of activity in cell extracts as a function of age, but separation by gel filtration on a Superose 6 column was found to be a useful method for determining the level of different proteasome related complexes.

Elevated SP-1 Transcription Factor Expression and Activity Drives Basal and Hypoxia Induced Vascular Endothelial Growth Factor (VEGF) Expression in Non-Small Cell Lung Cancer

Background: VEGF is central to cancer angiogenesis; however, we have a poor understanding of how VEGF is regulated in lung tumors. Results: High levels of SP-1 transcription factor expression amplify basal and hypoxia-induced VEGF expression. Conclusion: SP-1 plays a key role in both genetic and hypoxic microenvironment regulation of VEGF in cancer. Significance: Targeting of both VEGF and SP-1 may provide a more effective cancer therapy. VEGF plays a central role in angiogenesis in cancer. Non-small cell lung cancer (NSCLC) tumors have increased microvascular density, localized hypoxia, and high VEGF expression levels; however, there is a lack of understanding of how oncogenic and tumor microenvironment changes such as hypoxia lead to greater VEGF expression in lung and other cancers. We show that NSCLC cells secreted higher levels of VEGF than normal airway epithelial cells. Actinomycin D inhibited all NSCLC VEGF secretion, and VEGF minimal promoter-luciferase reporter constructs were constitutively active until the last 85 base pairs before the transcription start site containing three SP-1 transcription factor-binding sites; mutation of these VEGF promoter SP-1-binding sites eliminated VEGF promoter activity. Furthermore, dominant negative SP-1, mithramycin A, and SP-1 shRNA decreased VEGF promoter activity, whereas overexpression of SP-1 increased VEGF promoter activity. Chromatin immunoprecipitation assays demonstrated SP-1, p300, and PCA/F histone acetyltransferase binding and histone H4 hyperacetylation at the VEGF promoter in NSCLC cells. Cultured NSCLC cells expressed higher levels of SP-1 protein than normal airway epithelial cells, and double-fluorescence immunohistochemistry showed a strong correlation between SP-1 and VEGF in human NSCLC tumors. In addition, hypoxia-driven VEGF expression in NSCLC cells was SP-1-dependent, with hypoxia increasing SP-1 activity and binding to the VEGF promoter. These studies are the first to demonstrate that overexpression of SP-1 plays a central role in hypoxia-induced VEGF secretion.

Antivector and tumor immune responses following adenovirus-directed enzyme prodrug therapy for the treatment of prostate cancer.

We have completed a phase I/II suicide gene therapy clinical trial in patients with prostate cancer, using an E1/E3-deleted replication-deficient adenovirus (CTL102) encoding the bacterial nitroreductase enzyme in combination with prodrug CB1954. This study has provided an opportunity to monitor and characterize vector- and tumor-specific adaptive immunity before and after single or repeat injections of adenovirus. Here we report robust vector-specific humoral and cellular immune responses in all patients monitored. However, we found no correlation between preexisting immunity or the magnitude of the immune response to vector and the clinical outcome as measured by changes in serum prostate-specific antigen (PSA) level. Increased frequency of T cells recognizing prostate-specific antigens PSA or prostate-specific membrane antigen (PSMA) was detected in 3 of 11 patients after therapy, suggesting that this direct cytotoxic strategy can also stimulate tumor-specific immunity.

3-Dimensional Patient-Derived Lung Cancer Assays Reveal Resistance to Standards-of-Care Promoted by Stromal cells but Sensitivity to Histone Deacetylase Inhibitors

There is a growing recognition that current preclinical models do not reflect the tumor microenvironment in cellular, biological, and biophysical content and this may have a profound effect on drug efficacy testing, especially in the era of molecular-targeted agents. Here, we describe a method to directly embed low-passage patient tumor–derived tissue into basement membrane extract, ensuring a low proportion of cell death to anoikis and growth complementation by coculture with patient-derived cancer-associated fibroblasts (CAF). A range of solid tumors proved amenable to growth and pharmacologic testing in this 3D assay. A study of 30 early-stage non–small cell lung cancer (NSCLC) specimens revealed high levels of de novo resistance to a large range of standard-of-care agents, while histone deacetylase (HDAC) inhibitors and their combination with antineoplastic drugs displayed high levels of efficacy. Increased resistance was seen in the presence of patient-derived CAFs for many agents, highlighting the utility of the assay for tumor microenvironment-educated drug testing. Standard-of-care agents showed similar responses in the 3D ex vivo and patient-matched in vivo models validating the 3D-Tumor Growth Assay (3D-TGA) as a high-throughput screen for close-to-patient tumors using significantly reduced animal numbers. Mol Cancer Ther; 15(4); 753–63. ©2016 AACR.

HLA-peptide multimer selection of adenovirus-specific T cells for adoptive T-cell therapy

Adenovirus (Ad) infection is a cause of significant morbidity and mortality in hematopoietic stem cell transplant recipients and virus-specific immunotherapy is one option for improved control. Cellular immunity is an important component in suppression of Ad replication but the frequency and population distribution of Ad-specific CD8+ T cells has not been systematically investigated. This is an important question in relation to the potential use of these cells for adoptive transfer. To address this question, HLA-peptide multimers were generated for 8 HLA class I-restricted Ad epitopes, which are highly conserved across Ad species. Epitope-specific CD8+ T cells from healthy donors were identified by tetramer staining and HLA class I A*01-restricted TDL peptide staining T cells were characterized in relation to frequency, phenotype, and function. The cells demonstrated a minimally differentiated central memory phenotype (CD45RAhigh, CD45ROhigh, CCR7high, CD62Llow, CD27high, CD28high, and CD57low) and were able to produce IFN-&ggr; and proliferate extensively upon antigen stimulation in vitro. After proliferation, the phenotype switched to CD45ROhigh, although it is interesting to note that CCR7 expression was retained. Despite their low frequency, tetramer-staining cells could be enriched with magnetic bead technology. Their characteristics should permit rapid establishment in vivo post adoptive transfer, increasing therapeutic options for patients with Ad infection. This is the first reported characterization of Ad-specific tetramer-staining T cells with a view to adoptive transfer to hematopoietic stem cell transplant patients with Ad infection. The efficacy of these cells needs to be further evaluated in the setting of a clinical trial.

C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

C-terminal-binding protein interacting protein (CtIP) was first isolated as a binding partner of C-terminal-binding protein (CtBP). It is considered to contribute to the transcriptional repression and cell cycle regulatory properties of the retinoblastoma (Rb) family of proteins and to have a role in the cellular response to DNA damage. Here, we have shown that CtIP is a novel target for the adenovirus oncoprotein early region 1A (AdE1A). AdE1A associates with CtIP in both Ad5E1-transformed cells and Ad5-infected cells and binds directly in glutathione-S-transferase pull-down assays. Two binding sites have been mapped on Ad5E1A – the N-terminal α-helical region (residues 1–30) and conserved region 3 (CR3) – the transcriptional activation domain. CtIP can bind AdE1A and CtBP independently, raising the possibility that ternary complexes exist in Ad-transformed and -infected cells. Significantly, reduction of CtIP expression with small interfering RNAs results in reduction of the ability of a Gal4 DNA-binding domain-CR3 construct to transactivate a Gal 4-responsive luciferase reporter and this effect is reversed by reduction of CtBP expression. Therefore, in this model, CtIP acts as a transcriptional co-activator of AdE1A when dissociated from CtBP, through the action of AdE1A. These data are consistent with observations that CtIP expression is induced by AdE1A during viral infection and that reduction of CtIP expression with RNA interference can retard virus replication. In addition, AdE1A causes disruption of the CtIP/Rb complex during viral infection by its interaction with CtIP, possibly contributing to transcriptional derepression.

Quantum Dot Labelling of Adenovirus Allows Highly Sensitive Single Cell Flow and Imaging Cytometry

A quantum dot method for highly efficient labelling of single adenoviral particles is developed. The technique has no impact on viral fitness and allows the imaging and tracking of virus binding and internalisation events using a variety of techniques including imaging cytometry and confocal microscopy. The method is applied to characterise the tropism of different adenoviral vectors.

Adenovirus vector-specific T cells demonstrate a unique memory phenotype with high proliferative potential and coexpression of CCR5 and integrin α4β7

Background: The Step Study was a randomized trial to reduce HIV infection through vaccination with an adenovirus type 5 (Ad5)-based gag/pol/nef construct; analysis following early cessation of the trial revealed an excess of HIV seroconversion in Ad5 seropositive men. This led to the suggestion that the Ad based vector may boost the number of CD4+ chemokine receptor 5 (CCR5+) T cells, target cells for HIV infection. Objectives: We sought to determine the immunophenotype and proliferative capacity of Ad5-specific T cells in the peripheral blood of adult donors to determine whether stimulation with replication defective Ad5 vectors could result in the significant expansion of a CD4+ CCR5+ T-cell subset. Methods: Ad5-specific T cells were identified in the peripheral blood of healthy donors by interferon-γ secretion assay and proliferative response was measured by carboxyfluorescein succinimidyl ester labelling. Cells were analyzed by flow cytometry to determine T-cell differentiation marker, CCR5 and α4β7 expression on memory and proliferated cells. Results: Ad5-specific CD4+ T cells within healthy adult donors exhibit a unique minimally differentiated memory phenotype with coexpression of CD45RA, CD45RO and CCR7. Stimulation with Ad vector leads to rapid expansion in vitro and a switch to an effector memory phenotype. Both short-term reactivated and proliferating Ad5-specific CD4+ T cells express the HIV coreceptor CCR5 and the HIV gp120-binding integrin α4β7. Conclusion: Ad5-specific T cells demonstrate a phenotype and proliferative potential that would support HIV infection; these results are pertinent to the findings of the Step Study and future use of Ad5 as a vaccine vector.

Adenovirus retargeting and systemic delivery.

The useful application of gene therapy strategies is frequently thwarted by inadequate systems for delivery and expression of therapeutic transgenes within target cell populations. This is particularly important in diseases, such as metastatic cancer, where the target cells are disseminated around the body at the time of diagnosis. Even where target issues are relatively localised, gaining adequate transgene expression can still limit the usefulness of the approach; indeed, the only situations where gene therapy has been applied with success relate to target cell populations (such as haematopoietic stem cells) that can be returned to the patient following transduction ex vivo, or where a significant proportion of target cells can be transduced following local injection of the vector. For the vast majority of potential therapies, inadequate transgene delivery and expression remain a major bottleneck to progress.