Delivery of a First-in-Class miR-10b Antagomir to Treat Metastatic Cancer:The Long Winding Road from Pre-Clinical Development to Initial Clinical Experience

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MicroRNA sensors

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.We have developed a technology for the profiling of miRNA expression in intact cells. The approach is based on sensor oligonucleotides, which upon entering the cell, bind specific miRNA targets, are cleaved as a result of this binding, and produce a fluorescent signal that is proportional to the abundance of the miRNA target. Specifically, the sensor oligonucleotides are completely complementary to a target miRNA species, are non-stabilized around the seed region (the region cleaved by the miRNA-RISC), and are labeled with a fluorescent dye and a quencher at their 5’- and 3’- end respectively. Upon entering the cell, these oligonucleotides engage the target miRNA by complementary base pairing. This leads to recruitment of the RNA induced silencing complex (RISC) to the duplex. The complex cleaves the sensor oligonucleotide and the miRNA is free to “catalyze” subsequent clevage reactions. The cleavage of the sensor oligo leads to separation between the dye and the quencher, and a resultant fluorescent enhancement that can be measured. We have demonstrated the feasibility of this method for the sensing of the pro-metastatic miRNA-10b in cell-free extracts and intact cells using human and murine breast adenocarcinoma cell lines. The miRNA epigenome represents a fundamental molecular regulator of metastasis. Consequently, developing tools to understand metastatic changes at the miRNA level can lead to the mapping out of a comprehensive and systematic atlas of cancer progression. The described technology is potentially transformative because it addresses this important issue. Furthermore, the technology has broad implications and can be utilized in any model system or clinical scenario to answer questions related to microRNA function. Specifically, the technology can help distinguish, assess, and/or monitor cancer stages and progression; aid the elucidation of basic mechanisms underlying cancer initiation and progression; facilitate early cancer detection and/or cancer risk assessment; and facilitate/accelerate the process of drug discovery

The effects of erythrocyte alloantigen L on the avian immune response

Experiment 1 examined the alloantigen system L effects on Rous sarcomas in three B complex genotypes. The parental stock were 50% Modified Wisconsin Line 3 and 50% inbred Line 6.15- 5. B2B5 L1L2 x B2B5 L 1L2 matings produced experimental chicks. Chicks were inoculated with 20 pock-forming units (pfu) Rous sarcoma virus (RSV) at 6 weeks-of-age. Tumors were scored six times over 10 weeks postinoculation. Tumor scores were used to assign a tumor profile index (TPI) to each chicken. Results were evaluated by ANOVA. The B complex affected the responses. Separate analyses revealed L system effects ( P \u3c 0.05) only in B5B 5 chickens. Experiment 2 examined the influence of Ea-L on antibody response to SRBC and Brucella abortus (BA). The mating protocol was the same as in Experiment 1. At 4 and 11 weeks of age the experimental birds were injected intravenously with standard concentrations of SRBC and BA. Total and ME-resistant antibody titers were determined as described. Results were analyzed by ANOVA. Ea-L had an effect on total primary antibody titer to SRBC in a B5B 5 background (p \u3c 0.004) and on total (p \u3c 0.011) and ME-resistant (p \u3c 0.017) secondary titer to SRBC in a B5 B5 genotypic background. Ea-L also affected total (p \u3c 0.004) and ME-resistant (p \u3c 0.005) secondary titer to Brucella abortus in a B5 B5 background. Experiment 3 examined the effect of Ea-L on resistance and acquired immunity to E. tenella infection. The mating protocol was the same as in Experiments 1 and 2. In the resistance and susceptibility study, chicks were weighed and inoculated with 30,000 E. tenella oocysts at 6 weeks of age. 6 days post-inoculation, the birds were weighed again and assigned a cecal lesion score. In the immunity study, the challenge procedure was preceded by inoculations of 500 oocysts per day beginning at 5 weeks. Weight gain and cecal lesion scores were evaluated by ANOVA. The B complex affected lesion score in the immunity but not the resistance and susceptibility study and did not affect weight gain in either study. The L system had no effect in either study

Combined Small Interfering RNA Therapy and In Vivo Magnetic Resonance Imaging in Islet Transplantation

OBJECTIVE Recent advances in human islet transplantation are hampered by significant graft loss shortly after transplantation and inability to follow islet fate directly. Both issues were addressed by utilizing a dual-purpose therapy/imaging small interfering RNA (siRNA)-nanoparticle probe targeting apoptotic-related gene caspase-3. We expect that treatment with the probe would result in significantly better survival of transplanted islets, which could be monitored by in vivo magnetic resonance imaging (MRI). RESEARCH DESIGN AND METHODS We synthesized a probe consisting of therapeutic (siRNA to human caspase-3) and imaging (magnetic iron oxide nanoparticles, MN) moieties. In vitro testing of the probe included serum starvation of the islets followed by treatment with the probe. Caspase-3 gene silencing and protein expression were determined by RT-PCR and Western blot, respectively. In vivo studies included serial MRI of NOD-SCID mice transplanted with MN-small interfering (si)Caspase-3–labeled human islets under the left kidney capsule and MN-treated islets under the right kidney capsule. RESULTS Treatment with MN-siCaspase-3 probe resulted in decrease of mRNA and protein expression in serum-starved islets compared with controls. In vivo MRI showed that there were significant differences in the relative volume change between MN-siCaspase-3–treated grafts and MN-labeled grafts. Histology revealed decreased caspase-3 expression and cell apoptosis in MN-siCaspase-3–treated grafts compared with the control side. CONCLUSIONS Our data show the feasibility of combining siRNA therapy and in vivo monitoring of transplanted islets in mice. We observed a protective effect of MN-siCaspase-3 in treated islets both in vitro and in vivo. This study could potentially aid in increasing the success of clinical islet transplantation

Iron oxide nanoparticles and derivatives for biomedical imaging and application in cancer diagnosis and siRNA therapy

This paper was presented at the 3rd Micro and Nano Flows Conference (MNF2011), which was held at the Makedonia Palace Hotel, Thessaloniki in Greece. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, Aristotle University of Thessaloniki, University of Thessaly, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute.Our studies have focused on the application of imaging-capable nanoparticulate agents for the delivery of small RNA-based tumor therapy. One example includes magnetic nanoparticles (MN), which have traditionally been utilized as contrast agents for magnetic resonance imaging. The probes typically consist of a dextran-coated superparamagnetic iron oxide core (for magnetic resonance imaging), labeled with Cy5.5 dye (for near-infrared in vivo optical imaging), and conjugated to synthetic small interfering RNA (siRNA) molecules targeting model or therapeutic genes. We have explored the potential of these nanoparticles as delivery modules for small interfering RNA to tumors. Furthermore, we have investigated the feasibility of combining the imaging and delivery capabilities of these nanoparticles for the tracking of siRNA bioavailability. The versatile functionalization potential of MN has allowed us to control properties of the agents, such as uptake mechanism and target organ distribution. The tumoral accumulation of MNsiRNA results in a remarkable level of target-gene down-regulation. Repeated treatment with MN-siBIRC5, targeting the tumor-specific anti-apoptotic gene, birc5, leads to the induction of apoptosis in the tumors and an overall reduction in tumor growth rate. More recently, we have synthesized a second generation of nanoparticles, which combine the capability for high-resolution magnetic resonance imaging with detection by ultrasensitive surface enhanced Raman scattering

In Vivo Detection of miRNA Expression in Tumors Using an Activatable Nanosensor

Purpose The development of tools for the analysis of microRNA (miRNA) function in tumors can advance our diagnostic and prognostic capabilities. Here, we describe the development of technology for the profiling of miRNA expression in the tumors of live animals. Procedures The approach is based on miRNA nanosensors consisting of sensor oligonucleotides conjugated to magnetic nanoparticles for systemic delivery. Feasibility was demonstrated for the detection of miR-10b, implicated in epithelial to mesenchymal transition and the development of metastasis. The miR-10b nanosensor was tested in vivo in two mouse models of cancer. In the first model, mice were implanted subcutaneously with MDA-MB-231-luc-D3H2LN tumors, in which miR-10b was inhibited. In the second model, mice were implanted bilaterally with metastatic MDA-MB-231 and nonmetastatic MCF-7 cells. The nanosensors were injected intravenously, and fluorescence intensity in the tumors was monitored over time. Results We showed that the described nanosensors are capable of discriminating between tumors based on their expression of miR-10b. Radiant efficiency was higher in the miR-10b-active tumors than in the miR-10b-inhibited tumors and in the MDA-MB-231 tumors relative to the MCF-7 tumors. Conclusions The described technology provides an important tool that could be used to answer questions about microRNA function in cancer.National Institutes of Health (U.S.) (National Cancer Institute R01CA16346101A1)Breast Cancer Alliance (Young Investigator Award

A Novel Imaging Approach for Early Detection of Prostate Cancer Based on Endogenous Zinc Sensing

The early detection of prostate cancer is a life-saving event in patients harboring potentially aggressive disease. With the development of malignancy, there is a dramatic reduction in the zinc content of prostate tissue associated with the inability of cancer cells to accumulate the ion. In the current study, we used endogenous zinc as an imaging biomarker for prostate cancer detection and progression monitoring. We employed a novel fluorescent sensor for mobile zinc (ZPP1) to detect and monitor the development of prostate cancer in a transgenic mouse model of prostate adenocarcinoma, using in vivo optical imaging correlated with biological fluid-based methods. We showed that the progression of prostate cancer could be monitored in vivo judging by the decreasing zinc content in the prostates of tumor-bearing mice in an age-dependent manner. In a novel quantitative assay, we determined the concentration of mobile zinc in both prostate cell lysates and mouse prostate extracts through simple titration of the ZPP1 sensor. Our findings fulfill the promise of zinc-based prostate cancer diagnostics with the prospect for immediate clinical translation.National Institute of General Medical Sciences (U.S.) (grant GM065519)National Cancer Institute (U.S.) (grant R00CA129070)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (grant RC1DK086242)Massachusetts Biomedical Research CorporationMassachusetts Biomedical Research Corporation (Tosteson Postdoctoral Fellowship

Predictive imaging of chemotherapeutic response in a transgenic mouse model of pancreatic cancer: Imaging of Chemotherapeutic Response

The underglycosylated mucin 1 tumor antigen (uMUC1) is a biomarker that forecasts the progression of adenocarcinomas. In this study, we evaluated the utility of a dual-modality molecular imaging approach based on targeting uMUC1 for monitoring chemotherapeutic response in a transgenic murine model of pancreatic cancer (KCM triple transgenic mice). An uMUC1-specific contrast agent (MN-EPPT) was synthesized for use with magnetic resonance imaging (MRI) and fluorescence optical imaging. It consisted of dextran-coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1-specific peptide (EPPT). KCM triple transgenic mice were given gemcitabine as chemotherapy while control animals received saline injections following the same schedule. Changes in uMUC1 levels following chemotherapy were monitored using T2-weighted MRI and optical imaging before and 24 hr after injection of the MN-EPPT. uMUC1 expression in tumors from both groups was evaluated by histology and qRT-PCR. We observed that the average delta-T2 in the gemcitabine-treated group was significantly reduced compared to the control group indicating lower accumulation of MN-EPPT, and correspondingly, a lower level of uMUC1 expression. In vivo optical imaging confirmed the MRI findings. Fluorescence microscopy of pancreatic tumor sections showed a lower level of uMUC1 expression in the gemcitabine-treated group compared to the control, which was confirmed by qRT-PCR. Our data proved that changes in uMUC1 expression after gemcitabine chemotherapy could be evaluated using MN-EPPT-enhanced in vivo MR and optical imaging. These results suggest that the uMUC1-targeted imaging approach could provide a useful tool for the predictive assessment of therapeutic response

CARS microscopy for the visualization of micrometer-sized iron oxide MRI contrast agents in living cells

Micrometer-sized iron oxide particles (MPIOs) attract increasing interest as contrast agents for cellular tracking by clinical Magnetic Resonance Imaging (MRI). Despite the great potential of MPIOs for in vivo imaging of labeled cells, little is known on the intracellular localization of these particles following uptake due to the lack of techniques with the ability to monitor the particle uptake in vivo at single-cell level. Here, we show that coherent anti-Stokes Raman scattering (CARS) microscopy enables non-invasive, label-free imaging of MPIOs in living cells with sub-micron resolution in three dimensions. CARS allows simultaneous visualization of the cell framework and the MPIOs, where the particles can be readily distinguished from other cellular components of comparable dimensions, and localized inside the cell