MicroRNA Controlled Adenovirus Mediates Anti-Cancer Efficacy without Affecting Endogenous MicroRNA Activity

MicroRNAs are small non-coding RNA molecules that regulate mRNA translation and stability by binding to complementary sequences usually within the 3′ un-translated region (UTR). We have previously shown that the hepatic toxicity caused by wild-type Adenovirus 5 (Ad5WT) in mice can be prevented by incorporating 4 binding sites for the liver-specific microRNA, mir122, into the 3′ UTR of E1A mRNA. This virus, termed Ad5mir122, is a promising virotherapy candidate and causes no obvious liver pathology. Herein we show that Ad5mir122 maintains wild-type lytic activity in cancer cells not expressing mir122 and assess any effects of possible mir122 depletion in host cells. Repeat administration of 2×1010 viral particles of Admir122 to HepG2 tumour bearing mice showed significant anti-cancer efficacy. RT-QPCR showed that E1A mRNA was down-regulated 29-fold in liver when compared to Ad5WT. Western blot for E1A confirmed that all protein variants were knocked down. RT-QPCR for mature mir122 in infected livers showed that quantity of mir122 remained unaffected. Genome wide mRNA microarray profiling of infected livers showed that although the transcript level of >3900 different mRNAs changed more than 2-fold following Ad5WT infection, less than 600 were changed by Ad5mir122. These were then filtered to select mRNAs that were only altered by Ad5mir122 and the remaining 21 mRNAs were compared to predicted mir122 targets. No mir122 target mRNAs were affected by Ad5 mir122. These results demonstrate that the exploitation of microRNA regulation to control virus replication does not necessarily affect the level of the microRNA or the endogenous mRNA targets

Exploiting pre-existing viral immunity for B cell vaccination against endogenous antigens

Cancer vaccines are a novel method of treating cancer by harnessing the patient’s own immune system to recognise and attack malignant cells. Viral vectors can be used to encode tumour antigens to be presented to the immune system. However this field is hindered by immunity to the viral vectors themselves, which are often more immunogenic than tumour antigens.This thesis describes the production of a novel cancer vaccine based on murine leukaemia retrovirus (MLV), where the enveloped vaccine particles express only tumour antigens on the surface with internal viral proteins physically shielded from B cells. This is designed to stimulate B cells specific for the weak tumour antigens by exploiting T cell recognition of strong viral MHC class II epitopes to confirm their activation.Having cloned tumour antigens HER2 and 5T4 into the genome of MLV, the recombinant virus particles were purified and characterised thoroughly to ensure correct expression and presentation of tumour antigens. A vaccination schedule was also optimised, involving a homologous prime-boost strategy. Several prime vaccines were trialled with different results, highlighting the importance of priming CD4+ T cells and avoiding off-target effects. In vivo testing of the vaccine particles in BALB/c mice showed that mice with pre-existing immunity to MLV (those that were “primed” beforehand) had higher antibody responses against the tumour antigen than non-primed mice. This supports the hypothesis that the presence of viral-specific T cells will amplify the immune response to the tumour antigen after the subsequent “boost” vaccination, based on the requirement for T cell help for B cell activation and antibody production.This thesis demonstrates that tolerance to endogenous tumour antigens can be overcome by using a self-adjuvanting viral vaccine particle. This method of homologous prime-boosting has important implications for cancer vaccination and immunotherapy.</p

MicroRNA mediated knockdown of both E1A mRNA and protein measured by RT-QPCR and western blot after intravenous injection of 2×10¹⁰ vp of Ad5mir122.

<p>A) RT QPCR for the 13S E1A mRNA transcript in the livers of mice 48 hrs after intravenous injection with 2×10¹⁰ vp of Ad5mir122, Ad5WT, Ad5Luc or PBS. Ad5mir122 shows significantly reduced E1A mRNA during liver infection when compared to Ad5WT (N = 3). Statistical analysis was performed using a two tailed student T-Test (*P = <0.05). B) Western blot to confirm that all E1A proteins variants are knocked down. Each lane represents protein extracted from an individual mouse. Control lanes containing liver from mice treated with either an E1A deleted Ad5Luc vector or PBS show no E1A signal. Ad5WT treatment shows 3 clearly defined bands corresponding to proteins produced from the 13S (36 kDa), 12S (26 kDa) and a smaller fainter band which may represent the 11S or the 10S E1A transcript product. Treatment with Admir122 shows significant knockdown in E1A protein levels for all splice variants. The blot was exposed for 1, 5 and 10 minutes with the 5 minute exposure presented here. Molecular weights were calculated against a dual colour molecular weight ladder (Bio-Rad).</p

Ad5mir122 shows reduced E1A activity in primary hepatocytes.

<p>Primary human hepatocytes were infected with either Ad5mir122luc or Ad5WTluc at 1 vp/cell. Luciferase levels are shown 3 days post infection (n = 3) as relative light units per mg total protein. Error bars represent standard deviation.</p

The level and activity of mature mir122 <i>in vivo</i> is unaffected by Ad5mir122.

<p>Mice were injected with 2×10¹⁰ vp of either Ad5mir122, Ad5WT, an E1A deleted Ad5luc vector or PBS. Quantification of mir122 mature RNA levels was performed using a Taqman microRNA assay specific for mir122. CT values were corrected against the levels of the microRNA, let7a, as a reference gene using the method published by Pfaffl M <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016152#pone.0016152-Pfaffl1" target="_blank">[25]</a>. A) RT-QPCR for mir122 showing nine superimposed amplification curves (from three mice) in each treatment group, before correction against let7a. Samples were reverse transcribed using equal amounts of total RNA (5 ng) and RT-PCR was performed using equal amounts of cDNA. CT values shown here represent the average of the reactions from three mice plus or minus standard deviation. B) The total number of mRNA changes recorded by genome wide mRNA profiling of extracted murine hepatic RNA. Positive signals are those in which the median mRNA level changed by ≧2-fold from all mice in each group in comparison to median mRNA level in mice treated with PBS (n = 3 for all groups). The total number of genes altered is calculated using the average of the three independent replicates and therefore no error bars are shown. C) Western blot analysis of the mir122 regulated protein Aldolase A in mice treated as above. Liver protein extracts were subjected to a BCA protein assay and equal loading was confirmed by Ponceau stain (data not shown).</p

Ad5mir122 kills mir122 negative cells with Ad5WT potency.

<p>Comparison of Ad5mir122 and Ad5WT in cancer cell lines incubated at a multiplicity of infection of 100 viral particles per cell. The percentage cell survival is shown 6 days post infection (N = 5) using an MTS cell survival assay. Statistical analysis was performed using one way ANOVA (* = p<0.05, NS  =  not significant).</p

Pharmacodynamics led dose escalation study to determine the optimal treatment dose with Ad5mir122 and Ad5WT in tumour bearing mice.

<p>A) Serum Alanine Transaminase (ALT) levels from mice receiving Ad5mir122, Ad5WT or PBS. ALT values for each group are shown at days 2, 5 and 8 (n = 3) after the first injection. Data is presented as ALT units per litre using the equation in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016152#s2" target="_blank">materials and methods</a>. B) Luciferase imaging 2 days after the first injection of Ad5mir122 (left panel) or Ad5WT (right panel). C) Luciferase imaging eight days after the first injection. Mice which received a single injection of Ad5WT are shown in the right panel and mice receiving three injections of Ad5mir122 are shown in the left panel. Images are all presented on the same scale. D) Dosage and treatment schedule for Ad5mir122 and Ad5WT. Viral doses are indicated above each line and include 10% of a Luciferase reporter virus (Ad5mir122luc). All mice were treated with bisphosphonate liposomes at day -1.</p

Family Resources, Gender, and Immigration: Changing Sources of Hong Kong Educational Inequality, 1971-2001-super-

This study gauged the impact of government-led educational expansion on Hong Kong's social stratification over a 30-year period. The historically close state control over school supply in Hong Kong allows us to test the effectiveness of public policy in changing the transmission of advantages across generations. Copyright (c) 2004 by the Southwestern Social Science Association.

Conditional Knockout of Hypoxia-Inducible Factor 1-Alpha in Tumor-Infiltrating Neutrophils Protects against Pancreatic Ductal Adenocarcinoma

Large numbers of neutrophils infiltrate tumors and comprise a notable component of the inflammatory tumor microenvironment. While it is established that tumor cells exhibit the Warburg effect for energy production, the contribution of the neutrophil metabolic state to tumorigenesis is unknown. Here, we investigated whether neutrophil infiltration and metabolic status promotes tumor progression in an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC). We observed a large increase in the proportion of neutrophils in the blood and tumor upon orthotopic transplantation. Intriguingly, these tumor-infiltrating neutrophils up-regulated glycolytic factors and hypoxia-inducible factor 1-alpha (HIF-1α) expression compared to neutrophils from the bone marrow and blood of the same mouse. This enhanced glycolytic signature was also observed in human PDAC tissue samples. Strikingly, neutrophil-specific deletion of HIF-1α (HIF-1αΔNφ) significantly reduced tumor burden and improved overall survival in orthotopic transplanted mice, by converting the pro-tumorigenic neutrophil phenotype to an anti-tumorigenic phenotype. This outcome was associated with elevated reactive oxygen species production and activated natural killer cells and CD8+ cytotoxic T cells compared to littermate control mice. These data suggest a role for HIF-1α in neutrophil metabolism, which could be exploited as a target for metabolic modulation in cancer

Prevalence and Characteristics of Chinese Patients With Duchenne and Becker Muscular Dystrophy

The aim of this collaborative study on Duchenne muscular dystrophy and Becker muscular dystrophy is to determine the prevalence and to develop data on such patients as a prelude to the development of registry in Hong Kong. Information on clinical and molecular findings, and patient care, was systematically collected in 2011 and 2012 from all Pediatric Neurology Units in Hong Kong. Ninety patients with dystrophinopathy were identified, and 83% has Duchenne muscular dystrophy. The overall prevalence of dystrophinopathy in Hong Kong in 2010 is 1.03 per 10 000 males aged 0 to 24 years. Among the Duchenne group, we observed a higher percentage (40.6%) of point mutations with a lower percentage (45.3%) of exon deletions in our patients when compared with overseas studies. Although we observed similar percentage of Duchenne group received scoliosis surgery, ventilation support, and cardiac treatment when compared with other countries, the percentage (25%) of steroid use is lower