Expression Of Firefly Luciferase Research Articles

Pulsed-wave low-dose ultrasound hyperthermia selectively enhances nanodrug delivery and improves antitumor efficacy for brain metastasis of breast cancer

The clinical application of chemotherapeutics for brain tumors remains a challenge due to limitation of blood-brain barrier/blood-tumor barrier (BBB/BTB). In this study, we investigated the effects of low-dose focused ultrasound hyperthermia (UH) on the delivery and therapeutic efficacy of pegylated liposomal doxorubicin (PLD) for brain metastasis of breast cancer. Murine breast cancer cells (4T1-luc2) expressing firefly luciferase were implanted into mouse striatum as a brain tumor model. The mice were intravenously injected with PLD with/without transcranial pulsed-wave/continuous-wave UH (pUH/cUH) treatment on day-6 after tumor implantation. pUH (frequency: 500kHz, PRF: 1000Hz, duty cycle: 50%) was conducted under equal acoustic power (2.2-Watt) and sonication duration (10-min) as cUH. The amounts of doxorubicin accumulated in the normal brain and tumor tissues were measured with fluorometry. The tumor growth responses for the control, pUH, PLD, PLD+cUH, and PLD+pUH groups were evaluated with IVIS. The PLD distribution and cell apoptosis were assessed with immunofluorescence staining. The results showed that pUH significantly enhanced the PLD delivery into brain tumors and the tumor growth was further inhibited by PLD+pUH without damaging the sonicated normal brain tissues. This indicates that low-dose transcranial pUH is a promising method to selectively enhance nanodrug delivery and improve the brain tumor treatment.

Production of infectious salmon anaemia virus (ISAV) ribonucleoprotein complexes using a mammalian cell based minigenome system

Developments in recombinant virus techniques have been crucial to understand the mechanisms of virulence acquisition and study the replication of many different negatively stranded RNA viruses. However, such technology has been lacking for infectious salmon anaemia virus (ISAV) until recently. This was due in part to the lack of a Polymerase I promoter in Atlantic salmon to drive the production of recombinant vRNA. Therefore, the present study investigated a different alternative to produce ISAV recombinant vRNA, based on Mouse Pol I promoter/terminator sequences and expression in baby hamster kidney (BHK-21) cells.As a first step, a pathogenic ISAV was demonstrated to replicate and produce viable virions in BHK-21 cells. This indicated that the virus could use the mammalian cellular and nuclear machinery to produce vRNA segments and viral proteins, albeit in a limited capacity.Co-transfection of vRNA expressing plasmids with cytomegalovirus (CMV) promoter constructs coding for the three viral polymerase and nucleoprotein led to the generation of functional ribonucleoproteins (RNPs) which expressed either, green fluorescence protein (GFP) or firefly luciferase (FF). Further experiments demonstrated that a 21h incubation at 37°C was optimal for RNPs production. Inhibition by ribavirin confirmed that FF expression was linked to specific RNPs polymerase transcription. The present minigenome system provides a novel and alternative approach to investigate various aspects of ISAV replication and potentially those of other negatively stranded RNA viruses. Expression of RNPs in mammalian cells could also provide a method for the rapid screening of anti-viral compounds targeting ISAV replication.

A multimodality imaging model to track viable breast cancer cells from single arrest to metastasis in the mouse brain.

Cellular MRI involves sensitive visualization of iron-labeled cells in vivo but cannot differentiate between dead and viable cells. Bioluminescence imaging (BLI) measures cellular viability, and thus we explored combining these tools to provide a more holistic view of metastatic cancer cell fate in mice. Human breast carcinoma cells stably expressing Firefly luciferase were loaded with iron particles, injected into the left ventricle, and BLI and MRI were performed on days 0, 8, 21 and 28. The number of brain MR signal voids (i.e., iron-loaded cells) on day 0 significantly correlated with BLI signal. Both BLI and MRI signals decreased from day 0 to day 8, indicating a loss of viable cells rather than a loss of iron label. Total brain MR tumour volume on day 28 also correlated with BLI signal. Overall, BLI complemented our sensitive cellular MRI technologies well, allowing us for the first time to screen animals for successful injections, and, in addition to MR measures of cell arrest and tumor burden, provided longitudinal measures of cancer cell viability in individual animals. We predict this novel multimodality molecular imaging framework will be useful for evaluating the efficacy of emerging anti-cancer drugs at different stages of the metastatic cascade.

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself. Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter.

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

Entry Sites of Venezuelan and Western Equine Encephalitis Viruses in the Mouse Central Nervous System following Peripheral Infection.

Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus; Togaviridae) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV.McM-fLUC and VEEV.3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed (Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis. VEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent.

Abstract A072: Reprogramming immune response with capsid-optimized AAV vectors for immunotherapy of cancer

Abstract In current studies we assessed the ability the of capsid-optimized adeno-associated virus (AAV) vectors expressing antigen to activate a specific T-cell response in vivo. We have showed that the efficacy of wild-type (WT) AAV vectors can be significantly enhanced by substituting critical serine (S) and threonine (T) residues on their capsids to valine (V). These residues were identified by analysis of the AAV capsid crystal structure and they can be recognized and phosphorylated by common serine/threonine cellular kinases such as JNK and p38 MAPK. Several different amino acids were tested and (V) was chosen because of the similarity of its structure with both (S) and (T), and lack of recognition by kinases. Thus, these modifications can prevent kinase-mediated phosphorylation of the AAV capsid, subsequent ubiquitination and proteasome-mediated degradation of the vectors. Next, the ovarian albumin (OVA), commonly used in mouse models as an antigen for immunization studies was used to evaluate ability of these vectors to initiate immune response. AAV6-WT-OVA and capsid-optimized AAV6-S662V+T492V-OVA vectors were injected intramuscularly in C57BL/6 mice with a dose of 5x10e11 vgs/mouse. Enchased green fluorescent protein (AAV6-EGFP) was used as negative control. In two weeks after injection, blood was collected and a number of OVA-specific T-cells were analyzed by stain with iTAg MHC Class I Murine Tetramer. The data suggest that the administration of AAV vectors expressing OVA led to a robust activation (approximately 9%) of specific T-cells compared to the mock and AAV-WT treated animals (less than 1%). Next, we evaluated the killing ability of these OVA specific T-cell isolated from splenocytes of C57BL/6 mice i.m. injected with AAV6-WT and AAV6-mutant vectors. Two-color fluorescence assay of cell-mediated cytotoxicity was used to estimate percentage of dead/alive target cells and generate a killing curve with different effectors to target cell ratio. Results of these experiments suggest that OVA-CD8 cells isolated from mice injected with AAV-S662V+T492V-OVA vectors have approximately 2-fold higher killing activity compared with OVA-CD8 cells from AAV-WT-OVA injected mice. Finally, we evaluated the ability of capsid-optimized AAV6 vectors to initiate a protective anti-cancer immune response. Prostatic acid phosphatase (PAP), a gene up regulated in both human and mouse prostate cancer, was used as a specific target. C57BL/6 mouse subcutaneously injected prostate cancer cell line, RM1, was used as an animal model. We genetically modified this cell line for a stable expression of firefly luciferase (FLuc) to monitor progression of tumor reduction or growth in live animals. First, animals were immunized with AAV6-S663V+T492-PAP or AAV6-WT-PAP vectors. Two weeks later mice were challenged with RM1-FLuc cells by subcutaneous injection. Results indicate suppression of tumor growth by AAV6-S663V+T492-PAP for approximately four weeks in comparison to one week and two weeks for negative control AAV6-GFP and AAV6-WT-PAP treated mice, respectively. In conclusion, successful inhibition of tumor growth in this artificial animal model would set the stage for potential clinical application. Citation Format: Munjal Pandy, Kellee Britt, George Aslanidi. Reprogramming immune response with capsid-optimized AAV vectors for immunotherapy of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A072.

HIV-1 Tat regulates the expression of the dcw operon and stimulates the proliferation of bacteria

Infections of pathogenic bacteria are very common in acquired immunodeficiency syndrome (AIDS) patients. However, the biological effects of HIV-1 Tat on bacteria are incompletely understood. In this study, HIV-1 Tat was expressed in Escherichia coli and Pseudomonas aeruginosa (PA01) to investigate its biological effects on bacteria. Bacterial cells expressing either HIV-1 Tat1-86 (Tat1-86) or HIV-1 Tat1-72 (Tat1-72) grow significantly faster than those with either only an empty vector or an unrelated control (GFP or Rluc). Supplementation of purified HIV-1 Tat1-86 or Tat1-101 protein into bacterial culture medium stimulated the growth of both E. coli and PA01. The expression profile of certain cell division-associated genes, such as those in the division cell wall (dcw) operon (ftsA, ftsQ, ftsW and ftsZ), yafO and zipA, was altered in HIV-1 Tat1-86 expressing E. coli BL21(DE3). Furthermore, the expression of firefly luciferase (Fluc) reporter gene, when engineered for control by the dcw promoter and terminator, was enhanced by HIV-1 Tat in E. coli, confirming that HIV-1 Tat transcriptionally regulates the expression of the dcw operon. The finding that HIV-1 Tat stimulates bacterial growth whether it is produced intracellularly or applied extracellularly may have relevance for HIV patients who are highly susceptible to opportunistic bacterial infections.Contents category: Viruses –Retroviruses.The GenBank accession number for the sequence of HIV-1 Tat1-86 is AF324439.1.

Stabilized plasmid-based system for bioluminescent labeling of multiple streptococcal species.

To determine if multiple streptococcal species can be easily labeled for biophotonic imaging using a toxin-antitoxin stabilized reporter plasmid containing the native firefly luciferase gene, originally developed for use in Group A Streptococcus. A number of streptococcal species including Group B Streptococcus, Group C Streptococcus, Group G Streptococcus, S. iniae, S. vestibularis, and S. salivarius were successfully transformed with the reporter plasmid. In absence of antibiotic selection, the plasmid had variable stability amongst the six strains. The expression of firefly luciferase was highest in Group B Streptococcus and S. iniae, as observed by the brightest signal and lowest detection limits in vitro. Multiple streptococcal species can be easily transformed with our toxin-antitoxin stabilized bioluminescent reporter plasmid. However, this plasmid shows variable stability and signal in different species, restricting its use for certain applications.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo.

221. The Utilization of Capsid-Optimized Adeno-Associated Virus (AAV) Vectors for Cancer Immunotherapy

Cancer, a leading cause of death in the human population today, is generally being treated with chemotherapeutic methods, which have adverse toxic side effects. Immunotherapy represents an attractive alternative to common treatment since it is based on activation of host immune system.In current studies we assessed the ability the of AAV vectors expressing antigen to activate a specific T-cell response in vivo. The ovarian albumin (OVA), commonly used in mouse models as an antigen for immunization studies was used to evaluate immune response. AAV6-WT-OVA and capsid-optimized AAV6-S662V+T492V-OVA vectors were injected intramuscularly in C57BL/6 mice with a dose of 5×10e11 vgs/mouse. Enchased green fluorescent protein (AAV6-EGFP) was used as negative control. In two weeks after injection, blood was collected and a number of OVA-specific T-cells were analyzed by stain with iTAg MHC Class I Murine Tetramer. The data shown suggest that the administration of AAV vectors expressing OVA led to a robust activation (approximately 9%) of specific T-cells compared to the mock and AAV-WT treated animals (less than 1%).Next, we evaluated the killing ability of these OVA specific T-cell. Splenocytes from C57BL/6 mice i.m. injected with AAV6-WT and AAV6-mutant vectors were isolated in 14 days after injection. OVA-CD8 cells were expanded in vitro in the presence of a predominant for C57BL/6 mice, SIINFEKL peptide. Stimulated T-cells were used for killing assay against mouse prostate cell line RM1 stably expressing OVA. Two-color fluorescence assay of cell-mediated cytotoxicity was used as described above to estimate percentage of dead/alive target cells and generate a killing curve with different effectors to target cell ratio. Results of these experiments suggest that OVA-CD8 cells isolated from mice injected with AAV-S662V+T492V-OVA vectors have higher killing activity compared with OVA-CD8 cells from AAV-WT-OVA injected mice. Control T-cell do not show significant cytotoxicity which eliminates the possibility of autoreactive response.Finally, we evaluated the ability of capsid-optimized AAV6 vectors to initiate a protective anti-cancer immune response. Prostatic acid phosphatase (PAP), a gene up regulated in both human and mouse prostate cancer, was used as a specific target. C57BL/6 mouse subcutaneously injected prostate cancer cell line, RM1, was used as an animal model. We genetically modified this cell line for a stable expression of firefly luciferase (FLuc) to monitor progression of tumor growth in live animals. First, animals were immunized with AAV6-S663V+T492-PAP or AAV6-WT-PAP vectors. Two weeks later mice were challenged with RM1-FLuc cells by subcutaneous injection. The effect of treatment on tumor growth was evaluated by whole body life imaging. Results indicate suppression of tumor growth by AAV6-S663V+T492-PAP for approximately four weeks in comparison to one week and two weeks for negative control AAV6-GFP and AAV6-WT-PAP treated mice, respectively.In conclusion, successful inhibition of tumor growth in this artificial animal model would set the stage for potential clinical application.

645. Improving RAAV Gene Expression Inflammatory Conditions Using Empty Capsids and an Immunosuppressive Compound

Objective: Gene therapy has the potential to treat rheumatic diseases. We have shown that rAAV5 is effective in delivering genes to the joint in vivo, however, the presence of macrophages in the inflamed joint might hampering AAV mediated gene delivery. To investigate this further, we determined if administration of agents that influence macrophage activity/number and/or addition of empty capsids had an effect on rAAV5-transgene expression in the joints of arthritic and healthy mice.Material and Methods: Expression of luciferase after i.a. injection of an rAAV5 vector expressing Fire-fly luciferase was investigated in mice with or without arthritis. Mice with collagen-induced arthritis (CIA) were injected with rAAV5.CMV.Fluc or NaCl in both knee joints on day 21 (unless indicated otherwise) and monitored for luciferase expression (from 3 days up till 6 months) by IVIS. Where indicated, empty capsids were co-administered with full particles at a 5:1 or 20:1 (empty: full) ratio. Macrophages were depleted or inhibited by administration of systemic clodronate liposomes (i.v.) or triamcinolone (i.m. or i.a.) 48 hrs prior to vector administration. Healthy mice were administered with vector and empty capsid.Results: We showed that in arthritic mice administration of rAAV5. CMV. Fluc after the onset of inflammation (d24) resulted in lower expression of luciferase compared to vector administration before onset of inflammation (d17). Both the addition of triamcinolone and clodronate improved luciferase expression over a period of 4 weeks (end of experiment). When empty capsids were added to full genome containing capsids in a 1:5 ratio, expression per joint, and the percentage of knee joints with a positive signal improved. A study to assess the duration of expression as well as to investigate the combination of these two approaches was performed. Similar to the previous study, we observed increased expression using triamcinolone or empty capsid alone, however the combination of macrophage inhibition and decoy capsids resulted in a synergistic enhancement of gene expression (5.85 fold, p = 0.001). This increase was sustained for 6 months post vector administration. As triamcinolone is usually administered directly into the inflamed joint of RA patients, we compared the efficacy of i.a. versus i.m. administration of triamcinolone and found that the enhancement of gene expression was independent of the route of administration. Next we wanted to see if empty capsid could enhance expression in non-inflamed joints. We observed increased expression in the presence of empty capsid, 4.8 fold at 5:1 ratio and up to 20 fold with the 20:1 ratio (p<0.05). In all experiments expression was limited to the knee joint region.Conclusions: We demonstrated that either depletion or inhibition of macrophages leads to an increase in gene expression. In addition, empty decoy capsids improve transgene expression in the presence or absence of inflammation. These data have implications for future applications of local gene therapy to the joint, or to other tissues that have an abundance of macrophages.

286. Systemic Delivery of AAV9-IFNβ Vectors for Glioblastoma Gene Therapy

Introduction: Glioblastoma multiforme (GBM) is the most prevalent primary brain tumor, with a median survival (MS) of only 14.6 months from diagnosis despite providing multimodal treatment including maximal surgical resection, radiation and chemotherapy. Due to the highly invasive properties of GBM cells, complete surgical resection is practically impossible, leading to tumor recurrence and death. Thus, new approaches are needed to prevent tumor recurrence.IFNb is a pleiotropic cytokine with potent anti-tumor effects. Previous studies have shown that local delivery of AAV-IFNb in a non-invasive GBM mouse model had a remarkable therapeutic effect. However local treatment may be insufficient to treat GBM where long distance migration is a common feature. Therefore the therapeutic approach should be matched to the tumor properties, which may be accomplished with a disperse therapeutic network generated by global brain gene delivery. Here we tested the therapeutic efficacy of an AAV9-IFNb vector infused via the tail vein in an orthotopic xenograft GBM model in athymic nude mice implanted in the striatum with 50,000 highly invasive GBM8 human glioblastoma cells stably expressing firefly luciferase (GBM8-fluc) for in vivo bioluminescence monitoring of tumor growth. AAV9-IFNb vector was infused via the tail vein at different times after tumor implantation.We performed a dose response study by infusing AAV9-IFNb vectors at different doses (1E11vg to 1E12vg/mouse) on d14 posttumor implantation. The improvement in survival with AAV9-IFNb was dose dependent and ranged from an increase in median survival of 111 days to complete tumor regression compared to 46 days in PBS-treated controls. Treatment with 3e11vg AAV9-IFNb resulted in the survival of 87.5% of animals for at least 8 months with no remaining evidence of GBM cells in the brain upon histological examination. This dose was considered the minimal effective dose (MED) and used in subsequent experiments. Next, we examined the impact of time between tumor implantation and treatment by infusion AAV9-IFNb at MED on d14, d21, d25 or d28. We found that d21 is the longest interval showing 100% long-term survival. Interestingly, the survival benefit was drastically reduced when treated on d25 or later, corroborated by Fluc imaging results and histological analysis showing untreated GBM growth accelerates considerably after d21.Finally, we tested the importance of global gene delivery in a model of multifocal GBM model with tumors implanted in both hemispheres. On d14, AAV9-IFNb was infused systemically at MED or injected intratumorally on one side of the brain. We found that systemic delivery resulted in reduction of tumor size in both hemispheres while intra-tumoral injection only impacted tumor growth on the injected side. All animals treated systemically with AAV9-IFNb survived long-term, while the median survival for animals receiving a unilateral injection of AAV9-IFNb was 57 days compared to 46 days for PBS-treated controls.Conclusion: Global delivery of AAV9-IFNb can be beneficial particularly to treat invasive or multifocal GBM and can provide long-term survival in the xenograft GBM model when treated before the onset of highly accelerated tumor growth.

111. Efficient Transduction in Neonatal Mice via Systemic Administration of an Adenovirus Vector

An adenovirus (Ad) vector is widely used as a gene delivery vector due to their various advantages. An Ad vector is applicable to innate genetic diseases, which are crucial targets of gene therapy. Patients of several innate genetic disorders often display symptoms in their early stage of life and are danger of death without any treatments. Radical treatments in infant ages are required in such patients. Gene therapy in neonates and infants is promising for such innate genetic disorders. In addition, although immune responses to an Ad vector hamper Ad vector-mediated transduction in adults, it is expected that such inhibitions do not occur in neonates and infants because of their immature immunity; however, transduction properties of Ad vectors in neonates and infants have not been fully examined. In this study, Ad vector-mediated transduction properties in neonatal mice were examined.A first-generation Ad vector containing a CMV promoter-driven firefly luciferase expression cassette was administered to newborn mice on the second day of life at a dose of 5.9×1011 infectious unit (IFU)/kg (this titer is equal to 1010 IFU/5-weeks old mice) via retro-orbital sinus. First, biodistribution of an Ad vector in neonatal mice was investigated 2 days after administration. As is the case with adult mice, the highest copy numbers of Ad vector genome were found in the liver among the organs. The liver also showed the highest levels of luciferase expression among the organs in neonates, although the transduction efficiency in the neonatal liver was approximately 5-fold lower than that in the adult liver. On the other hand, neonatal mice exhibited 10-fold higher levels of luciferase expression in the heart than adult mice. While approximately more than 1000-fold higher transduction efficiency was found in the adult liver, compared with the adult heart, the neonatal liver exhibited only 30-fold higher transduction efficiency than the neonatal heart. In order to achieve liver-specific transduction, a liver-specific promoter (a fusion promoter composed of apolipoprotein E enhancer, the hepatocyte control region, and human alpha 1-antitrypsin promoter) was incorporated into an Ad vector. The Ad vector containing the liver-specific promoter mediated almost 100-fold higher transduction efficiencies in the liver, compared with those in the other organs. Second, Ad vector-induced hepatotoxicity in neonatal mice was evaluated. Obvious elevation in serum alanine aminotransferase (ALT) activity in neonatal mice was not observed within 2 weeks following Ad vector administration, indicating that this dose of Ad vectors did not provoke significant hepatotoxicity in neonatal mice. In addition, while anti-Ad antibodies were strongly induced in the serum of adult mice on 21 days post Ad vector injection, almost background levels of anti-Ad antibodies were detected in neonatal mice. These results provide crucial information for gene therapy in neonates and infants.

Intra-adrenal murine TH-MYCN neuroblastoma tumors grow more aggressive and exhibit a distinct tumor microenvironment relative to their subcutaneous equivalents.

In around half of the patients with neuroblastoma (NBL), the primary tumor is located in one of the adrenal glands. We have previously reported on a transplantable TH-MYCN model of subcutaneous (SC) growing NBL in C57Bl/6 mice for immunological studies. In this report, we describe an orthotopic TH-MYCN transplantable model where the tumor cells were injected intra-adrenally (IA) by microsurgery. Strikingly, 9464D cells grew out much faster in IA tumors compared to the subcutis. Tumors were infiltrated by equal numbers of lymphocytes and myeloid cells. Within the myeloid cell population, however, tumor-infiltrating macrophages were more abundant in IA tumors compared to SC tumors and expressed lower levels of MHC class II, indicative of a more immunosuppressive phenotype. Using 9464D cells stably expressing firefly luciferase, enhanced IA tumor growth could be confirmed using bioluminescence. Collectively, these data show that the orthotopic IA localization of TH-MYCN cells impacts the NBL tumor microenvironment, resulting in a more stringent NBL model to study novel immunotherapeutic approaches for NBL.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1663-y) contains supplementary material, which is available to authorized users.

An Intergenic SNP in the β–globin Gene Cluster Is Associated with Hyperuricemia and Influences Gene Transcription in Vitro

Hemolysis as a suggested cause of hyperuricemia is based upon the fact that red blood cells contain uric acid. The development of hyperuricemia also generates from an increased synthesis of nucleic acids occurring as part of the erythropoietic response to hemolysis in hemoglobin disorders such as sickle cell anemia, α-thalassemia, and β-thalassemia. In addition, multiple genome-wide association studies (GWAS) have reported significant association between uric acid levels and specific genomic loci. However, the mechanism of hyperuricemia still remains controversial and it is also unknown whether African Americans have higher prevalence of hyperuricemia due to genetic vs environmental risk factors. Here, we used joint admixture mapping and association testing to identify genetic variants associated with serum uric acid levels in African American. Interestingly, we detected 6 SNPs (rs2855125, rs2855126, rs11036415, rs11036496, rs4320977, and rs4348933) in an intergenic region of the β–globin cluster on chromosome 11 are associated with high levels of serum uric acid in populations of African ancestry. Next, we explored the potential regulatory role of intergenic SNPs associated with hyperuricemia using luciferase reporter gene activity assays and electrophoretic mobility shift assays (EMSA). Each SNP-containing DNA fragment was amplified by PCR using human genomic DNA and inserted into a firefly luciferase reporter vector, pGL3-basic vector. 293T or K562 cells were co-transfected with these constructs and a Renilla luciferase vector to control for transfection efficiency. Expression of firefly luciferase driven by each SNP-containing DNA fragment was measured by a dual luciferase reporter assay and normalized by Renilla luciferase expression. SNPs rs2855126, rs11036496 and rs4348933 on chromosome 11 had significantly greater expression levels of firefly luciferase than pGL3-basic-transfected cells in both 293T and K562 cells. Of these, the SNP rs2855126 ancestral C allele (associated with higher serum uric acid levels) showed significantly higher luciferase activity than the derived G allele. Functionally, the luciferase activities from these constructs were determined to be very similar in both cell lines used. Alleles altering expression were further assessed for binding of nuclear extracted proteins by EMSA. We found specific gel shift bands for SNPs rs2855126, rs11036496, and rs4348933, suggesting these SNPs are situated in the binding site of potential transcription factors. These data provide new insights into the potential contribution of imbalanced β-globin gene expression to hyperuricemia. DisclosuresNo relevant conflicts of interest to declare.

Abstract 816: Antitumor efficacy of a novel anthracycline derivative aldoxorubicin in an orthotopic mouse model of glioblastoma

Abstract Background: Glioblastoma multiforme (GBM) is the most common and most malignant of all primary brain tumors with a median survival of only 12-15 months despite standard-of-care, including surgical resection, radiotherapy, and temozolomide. In this study, we evaluated the preclinical efficacy of a novel albumin-binding prodrug of doxorubicin, aldoxorubicin, for the treatment of GBM in a murine model, and compared its antitumor effect with doxorubicin. Methods: U87MG human glioma cells (5 x 105) expressing firefly luciferase (U87-luc) were stereotactically injected into the left striatum of nude mice. After 12 days, mice (n=8 in each group) received either vehicle or a single intravenous injection of 24 mg/kg [3/4 maximum tolerated dose (MTD)] aldoxorubicin, or 6 mg/kg (3/4 MTD) of doxorubicin once a week. Tumor growth was monitored weekly using in vivo quantitative bioluminescence imaging (photons/sec), and survival compared using Kaplan-Meier curves. Aldoxorubicin concentrations in the tumors and uninvolved brain tissues harvested from these mice were quantified by HPLC. Results: All animals in the control and doxorubicin groups developed large tumors (median tumor size 5.0 x 108 photons/sec) by day 22 and died within 34 days after tumor implantation. During the same period, animals treated with aldoxorubicin displayed significant tumor regression (median tumor size 1.6 x 108 photons/sec) and remained alive with a median survival of more than 60 days. Fluorescence microscopy showed selective accumulation of aldoxorubicin, but not doxorubicin, in the tumor tissues resected from tumor-bearing mice 24h following intravenous injection of these drugs. HPLC analysis revealed 3- to 4-fold higher aldoxorubicin retention in the tumor tissues than in the surrounding brain tissues. Immunohistochemical evaluation of aldoxorubicin-treated tumors showed that the drug significantly decreased the number of dividing cells stained with the nuclear-localized Ki67 proliferation marker, and activated the apoptosis effector cleaved caspase-3. Conclusion: Our preliminary findings indicate that aldoxorubicin, but not doxorubicin, administered intravenously induces tumor regression and significantly increases survival in an in vivo xenograft tumor model employing intracranial injection and growth of human GBM cells. Preferential accumulation and prolonged retention in the tumor tissues, combined with its safety profile, provide significant rationale for the assessment of aldoxorubicin in the treatment of patients with GBM tumors. Citation Format: Om Prakash, Albero E. Musto, Dorota Wyczechowska, Luis Marrero, Adriana Zapata, Chelsey P. Walker, Christopher Parsons, Scott Wieland, Daniel Levitt, Krzysztof Reiss. Antitumor efficacy of a novel anthracycline derivative aldoxorubicin in an orthotopic mouse model of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 816. doi:10.1158/1538-7445.AM2014-816

Abstract 707: Tumor-targeted delivery of siRNA using stabilized calcium phosphate nanoparticles based on bio-inspired hyaluronic acid conjugate

Abstract Purpose : Low cytotoxicity and high cellular gene delivery capability are among the most important prerequisites for the selection of a non-viral carrier. In this study, we report the development of calcium phosphate (CAP) nanoparticles stabilized by a conjugate of the biological adhesive molecule, 3,4-dihydroxy-L-phenylalanine (dopa), and a nontoxic hydrophilic natural polymer, hyaluronic acid (HA), for targeted siRNA delivery to cancer cells. Methods : Dopa-conjugated HA (dopa-HA) was synthesized by EDC/NHS chemistry and analyzed by spectrometry and 1H-NMR. Stabilized calcium phosphate nanoparticles containing siRNA (CAP/siRNA/dopa-HA) was prepared by simply mixing CaCl2 solution containing siRNA with a HEPES buffer containing Na2HPO4 and subsequent addition of a solution containing dopa-HA. The particles were characterized by using light scattering method and transmission electron microscope (TEM). The gene silencing and cancer cell-targeting ability of CAP/siRNA/dopa-HA was evaluated by monitoring cellular reporter activity (luciferase) in human colon carcinoma cells permanently expressing firefly luciferase. Confocal microscopy and FACS analysis were performed to assess CD44-specific cellular delivery. Bio-distribution and in vivo gene silencing of CAP/siRNA/dopa-HA nanoparticles were studied in an animal model bearing tumor xenograft. Results : CAP/siRNA/dopa-HA can form compact nanoparticles that effectively protect siRNA from enzymatic degradation despite the structural drawbacks of siRNA, such as low charge density and short and rigid structure. In addition, stabilized CAP nanoparticles were able to maintain their colloidal stability in a physiological salt condition for over a week. The superior ability of CAP/siRNA/dopa-HA to maintain the integrity of encapsulated siRNA and the stability in solution of the nanoparticles allow this formulation to achieve improved intratumoral accumulation of siRNA and a high level of target gene silencing in solid tumors after systemic administration. Conclusion Considering its biocompatibility, transfection efficacy, and tumor targeting capability, this stabilized organic-inorganic hybrid gene delivery platform should be considered a promising candidate carrier for systemic siRNA delivery and targeted cancer therapy. Citation Format: Min Sang Lee, Jung Eun Lee, Eunkyoung Byun, Nak Won Kim, Haeshin Lee, Ji Hoon Jeong. Tumor-targeted delivery of siRNA using stabilized calcium phosphate nanoparticles based on bio-inspired hyaluronic acid conjugate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 707. doi:10.1158/1538-7445.AM2014-707

Cardiac tissue slice transplantation as a model to assess tissue-engineered graft thickness, survival, and function.

Cell therapies offer the potential to improve cardiac function after myocardial infarction. Although injection of single-cell suspensions has proven safe, cell retention and survival rates are low. Tissue-engineered grafts allow cell delivery with minimal initial cell loss and mechanical support to the heart. However, graft performance cannot be easily compared, and optimal construct thickness, vascularization, and survival kinetics are unknown. Cardiac tissue slices (CTS) were generated by sectioning mouse hearts (n=40) expressing firefly luciferase and green fluorescent protein into slices of defined size and thickness using a vibrating blade microtome. Bioluminescence imaging of CTS transplanted onto hearts of immunodeficient mice demonstrated survival of ≤30% of transplanted cells. Cardiac slice perfusion was re-established within 3 days, likely through anastomosis of pre-existing vessels with the host vasculature and invasion of vessels from the host. Immunofluorescence showed a peak in cell death 3 days after transplantation and a gradual decline thereafter. MRI revealed preservation of contractile function and an improved ejection fraction 1 month after transplantation of CTS (28±2% CTS versus 22±2% control; P=0.05). Importantly, this effect was specific to CTS because transplantation of skeletal muscle tissue slices led to faster dilative remodeling and higher animal mortality. In summary, this is the first study to use CTS as a benchmark to validate and model tissue-engineered graft studies. CTS transplantation improved cell survival, established reperfusion, and enhanced cardiac function after myocardial infarction. These findings also confirm that dilative remodeling can be attenuated by topical transplantation of CTS but not skeletal muscle tissue grafts.

Hsa-miR-195 targets PCMT1 in hepatocellular carcinoma that increases tumor life span.

MicroRNAs are small 19-25 nucleotides which have been shown to play important roles in the regulation of gene expression in many organisms. Downregulation or accumulation of miRNAs implies either tumor suppression or oncogenic activation. In this study, differentially expressed hsa-miR-195 in hepatocellular carcinoma (HCC) was identified and analyzed. The prediction was done using a consensus approach of tools. The validation steps were done at two different levels in silico and in vitro. FGF7, GHR, PCMT1, CITED2, PEX5, PEX13, NOVA1, AXIN2, and TSPYL2 were detected with high significant (P < 0.005). These genes are involved in important pathways in cancer like MAPK signaling pathway, Jak-STAT signaling pathways, regulation of actin cytoskeleton, angiogenesis, Wnt signaling pathway, and TGF-beta signaling pathway. In vitro target validation was done for protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1). The co-transfection of pmirGLO-PCMT1 and pEGP-miR-195 showed highly significant results. Firefly luciferase was detected using Lumiscensor and t test analysis was done. Firefly luciferase expression was significantly decreased (P < 0.001) in comparison to the control. The low expression of firefly luciferase validates the method of target prediction that we used in this work by working on PCMT1 as a target for miR-195. Furthermore, the rest of the predicted genes are suspected to be real targets for hsa-miR-195. These target genes control almost all the hallmarks of liver cancer which can be used as therapeutic targets in cancer treatment.