Absence Of Induction Research Articles

Optimization of a DiCre recombinase system with reduced leakage for conditional genome editing of Cryptosporidium

BackgroundThe dimerizable Cre recombinase system (DiCre) exhibits increased leaky activity in Cryptosporidium, leading to unintended gene editing in the absence of induction. Therefore, optimization of the current DiCre technique is necessary for functional studies of essential Cryptosporidium genes.MethodsBased on the results of transcriptomic analysis of Cryptosporidium parvum stages, seven promoters with different transcriptional capabilities were screened to drive the expression of Cre fragments (FKBP-Cre59 and FRB-Cre60). Transient transfection was performed to assess the effect of promoter strength on leakage activity. In vitro and in vivo experiments were performed to evaluate the leaky activity and cleavage efficiency of the optimized DiCre system by polymerase chain reaction (PCR), nanoluciferase, and fluorescence analyses.ResultsThe use of promoters with lower transcriptional activity, such as pcgd6_4110 and pcgd3_260, as opposed to strong promoters such as pActin, pα-Tubulin, and pEnolase, reduced the leakage rate of the system from 35–75% to nearly undetectable levels, as verified by transient transfection. Subsequent in vitro and in vivo experiments using stable lines further demonstrated that the optimized DiCre system had no detectable leaky activity. The system achieved 71% cleavage efficiency in vitro. In mice, a single dose of the inducer resulted in a 10% conditional gene knockout and fluorescent protein expression in oocysts. These fluorescently tagged transgenic oocysts could be enriched by flow sorting for further infection studies.ConclusionsA DiCre conditional gene knockout system for Cryptosporidium with good cleavage efficiency and reduced leaky activity has been successfully established.Graphical

Assessing cell lines with inducible depletion of cohesin and condensins components through analysis of metaphase chromosome morphology.

One of the most productive strategies for finding the functions of proteins is to study the consequences of loss of protein function. For this purpose, cells or organisms with a knockout of the gene encoding the protein of interest are obtained. However, many proteins perform important functions and cells or organisms could suddenly lose fitness when the function of a protein is lost. For such proteins, the most productive strategy is to use inducible protein degradation systems. A system of auxin-dependent protein degradation is often implemented. To use this system, it is sufficient to introduce a transgene encoding a plant-derived auxin-dependent ubiquitin ligase into mammalian cells and insert a sequence encoding a degron domain into the gene of interest. A crucial aspect of development of cell lines engineered for inducible protein depletion is the selection of cell clones with efficient auxin-dependent degradation of the protein of interest. To select clones induced by depletion of the architectural chromatin proteins RAD21 (a component of the cohesin complex) and SMC2 (a component of the condensin complex), we propose to use the morphology of metaphase chromosomes as a convenient functional test. In this work, we obtained a series of clones of human HAP1 cells carrying the necessary genetic constructs for inducible depletion of RAD21 and SMC2. The degradation efficiency of the protein of interest was assessed by flow cytometry, Western blotting and metaphase chromosome morphology test. Based on our tests, we showed that the clones we established with the SMC2 degron effectively and completely lose protein function when induced by auxin. However, none of the HAP1 clones we created with the RAD21 degron showed complete loss of RAD21 function upon induction of degradation by auxin. In addition, some clones showed evidence of loss of RAD21 function even in the absence of induction. The chromosome morphology test turned out to be a convenient and informative method for clone selection. The results of this test are in good agreement with flow cytometry analysis and Western blotting data.

Differential gene expression of Wharton's jelly-derived mesenchymal cells mediated by graphene oxide in basal and osteo-induced media.

Graphene oxide (GO) is widespread in scaffold engineering owing to its extraordinary properties such as multiple oxygen functional groups, high hydrophilicity ability and biocompatibility. It is known to promote differentiation in mesenchymal stem cells, but concomitant comparison of its modulation on the expression profiles of Wharton's jelly (WJ)-MSC surface markers, lineage differentiation, and epigenetic regulatory genes in basal and induced condition are still lacking. Unraveling the fundamental mechanisms is essential for the effective utilization of WJ-MSCs incorporated with GO in therapy. This study aims to explore the unique gene expression profiles and epigenetic characteristics of WJ-MSCs influenced by GO. The characterized GO-coated coverslip served as a substrate for culturing WJ-MSCs. In addition to investigating the impact of GO on cell proliferation and differentiation, we conducted a gene expression study using PCR array, while epigenetic control was assessed through bisulfite sequencing and Western blot analysis. Our findings indicate that the presence of GO maintained the proliferation and survival of WJ-MSCs. In the absence of induction, GO led to minor lipid and glycosaminoglycan deposition in WJ-MSCs. This was evidenced by the sustained expression of pluripotency and lineage-specific genes, demethylation at the OCT4 promoter, and a decrease in H3K9 methylation. In osteo-induced condition, the occurrence of osteogenesis appeared to be guided by BMP/TGF and ERK pathway activation, accompanied by the upregulation of osteogenic-related genes and downregulation of DNMT3b. GO in osteo-induced condition create a favorable microenvironment that promotes the osteogenesis of WJ-MSCs by influencing genetic and epigenetic controls. This helps in advancing our knowledge on the use of GO as priming platform and WJ-MSCs an alternate source for bone repair and regeneration.

Improving the Z3EV promoter system to create the strongest yeast promoter.

Promoters for artificial control of gene expression are central tools in genetic engineering. In the budding yeast Saccharomyces cerevisiae, a variety of constitutive and controllable promoters with different strengths have been constructed using endogenous gene promoters, synthetic transcription factors and their binding sequences, and artificial sequences. However, there have been no attempts to construct the highest strength promoter in yeast cells. In this study, by incrementally increasing the binding sequences of the synthetic transcription factor Z3EV, we were able to construct a promoter (P36) with ~1.4 times the strength of the TDH3 promoter. This is stronger than any previously reported promoter. Although the P36 promoter exhibits some leakage in the absence of induction, the expression induction by estradiol is maintained. When combined with a multicopy plasmid, it can express up to ~50% of total protein as a heterologous protein. This promoter system can be used to gain knowledge about the cell physiology resulting from the ultimate overexpression of excess proteins and is expected to be a useful tool for heterologous protein expression in yeast.

Scarless DNA Recombineering.

The method described here allows editing of the bacterial genome without leaving any secondary changes (scars) behind. This method uses a tripartite selectable and counterselectable cassette comprising an antibiotic-resistance gene (cat or kan) and the tetR repressor gene linked to a Ptet promoter-ccdB toxin gene fusion. In the absence of induction, the tetR gene product represses the Ptet promoter, preventing ccdB expression. The cassette is first inserted at the target site by selecting for chloramphenicol or kanamycin resistance. It is subsequently replaced by the sequence of interest by selecting for growth in the presence of anhydrotetracycline (AHTc), which inactivates the TetR repressor thereby causing CcdB-induced lethality. Unlike other CcdB-based counterselection schemes, which require specifically designed λ-Red delivery plasmids, the system described here uses the popular plasmid pKD46 as the source of λ-Red functions. This protocol allows a wide variety of modifications, including the intragenic insertion of fluorescent or epitope tags, gene replacements, deletions, and single base-pair substitutions, to be made. In addition, the procedure can be used to place the inducible Ptet promoter at a chosen position in the bacterial chromosome.

Macrophage differentiation is marked by increased abundance of the mRNA 3' end processing machinery, altered poly(A) site usage, and sensitivity to the level of CstF64.

Regulation of mRNA polyadenylation is important for response to external signals and differentiation in several cell types, and results in mRNA isoforms that vary in the amount of coding sequence or 3' UTR regulatory elements. However, its role in differentiation of monocytes to macrophages has not been investigated. Macrophages are key effectors of the innate immune system that help control infection and promote tissue-repair. However, overactivity of macrophages contributes to pathogenesis of many diseases. In this study, we show that macrophage differentiation is characterized by shortening and lengthening of mRNAs in relevant cellular pathways. The cleavage/polyadenylation (C/P) proteins increase during differentiation, suggesting a possible mechanism for the observed changes in poly(A) site usage. This was surprising since higher C/P protein levels correlate with higher proliferation rates in other systems, but monocytes stop dividing after induction of differentiation. Depletion of CstF64, a C/P protein and known regulator of polyadenylation efficiency, delayed macrophage marker expression, cell cycle exit, attachment, and acquisition of structural complexity, and impeded shortening of mRNAs with functions relevant to macrophage biology. Conversely, CstF64 overexpression increased use of promoter-proximal poly(A) sites and caused the appearance of differentiated phenotypes in the absence of induction. Our findings indicate that regulation of polyadenylation plays an important role in macrophage differentiation.

316.1: Incidence and Effect on Survival of Delayed Graft Function in Donor-Deceased Kidney Transplants in Argentina: A Multicenter Analysis

Introduction: Delayed graft function (DGF) is a manifestation of acute kidney injury with attributes unique to the transplant process. DGF is associated with negative outcomes such as higher incidence of acute rejection, worse graft function and lower grafts and patients´ survival. Although the worldwide incidence of DGF is variable, a previous study showed that the incidence in Argentina is higher than the US and most countries in Europe. Therefore, the aim of our study was to identify the risk factors associated with DGF in Argentina and to evaluate the impact of DGF on grafts´ and patients´ survival. Methods: Retrospective multicenter cohort study of deceased-donor kidney transplant from 33 transplant centers in Argentina between 2015 and 2017. Demographic data and donor and recipient related variables were analyzed at baseline and at 3 and 5 years of follow-up. Data were obtained from SINTRA/ INCUCAI, the Argentinian procurement organization and from the transplant centers. Results: Between 2015 and 2017, 1886 single kidney transplants were performed in 33 centers. Recipient and donor characteristics are shown in Table 1. In the current study, the incidence of DGF was 62%. Univariate analysis demonstrated that higher donor creatinine, donor age, recipient time on dialysis, donor BMI, absence of induction and cold ischemia time were risk factors associated with DGF. Multiple organ donation and cause of death other than stroke were associated with reduced risk DGF. In the multivariable analysis donor age (0.02), higher donor creatinine (0.02), recipient time on dialysis (<0.01), cold ischemia time (<0.01), monorganic donor (<0.01)) were risk factors of DGF. Renal function was significatively better in patients without DGF throughout the 5 years follow -up (p<0.05). Patients with DGF had higher incidence of acute rejection in the first three months of follow-up, with T-cell mediated rejection being the most frequent. At 3 and 5 years of follow up there was no increased risk of rejection when both groups were compared. Graft survival and patient survival were lower in the group that developed DGF at 1, 3 and 5 years of follow-up (<0.01) (Figure 1). Conclusion: We confirmed the high incidence of DGF in our country. Furthermore, the analysis on the risk factors associated with DGF were not as much as different as in other countries where the incidence of DGF were lower. Further studies should be done to unravel the cause of this high incidence of DGF in Argentina. Heads of Transplant Centers: WalterJ, Raffaele P, Ciappa M, Paladini J, De la Fuente J, Novoa P, Soler Pujol G, Toselli L, Suso E, Ferreyra M, Fragale G, Reinoso S, Acosta F, Sgrosso J, Arriola M, Maldonado R, Acosta M, Prado R, Sedevich A, Martinez I, Di Pietrantonio S, Oddino J, Trimarchi H, Raimondi C, Gómez M, Dorado E, Palti G, Mele P, Maggiora E, Papaginovic Leiva M. Kidney Commitee: Ibañez J, Benitez ML, Leon L, Mesias M, Osella F.

Primum non Nocere: Induction in Kidney Transplantation After Heart Transplantation.

Kidney allograft transplantation after heart transplantation remains a rare event with 70 reported in 2007 and this number declining to 30 subsequently and more recently rose to 60 per year. The fact that renal allograft transplantation is needed at all is due to the well-known nephrotoxicity of calcineurin inhibitors, the mainstay of contemporary immunosuppression in cardiac transplant recipients.1 End-stage renal failure has increased significantly in nonrenal transplant recipients from 1998 to 2007.2 The Registry of the International Society of Heart and Lung Transplantation has reported that by 5 y posttransplant, 4.1% of heart transplant recipients develop end-stage renal disease requiring renal replacement strategies such as dialysis or renal transplantation and these percentages double by 10 y posttransplant.3 As survival after cardiac transplantation improves and patients are living longer, the number of patients requiring renal transplantation will be increasing, explaining the increase mentioned above. That these numbers have not been increasing more rapidly is a result of more assiduous attention to posttransplant management with mitigation of renal dysfunction by reduction in calcineurin inhibitor (CNI) target trough levels. This is a consequence of the study by Ojo et al.1 However, given the improving, long-term survival after heart transplantation resulting in longer-term exposure to CNIs, there will be more patients needing renal transplantation. Therefore, the identification of optimal immunosuppressive regimens guaranteeing successful outcomes in heart transplant recipients receiving renal transplants after their heart transplants as a consequence of CNI toxicity is important. Pre–heart transplant congestion and decreased cardiac output can impair renal function as well. Thus, the study by Riad et al4 published in this edition of Transplantation is important and novel. The authors reviewed the Scientific Registry of Transplant Recipient data from the United States of heart transplant recipients who subsequently, at a later date as a result of end-stage renal disease, underwent kidney transplantation (N=617) and compared post–kidney transplant outcomes as a function of type of induction or absence of induction in the early posttransplant period: depletional induction (ie, antilymphocyte globulin or alemtuzumab) (N = 307, 49.8%), nondepletional (ie, anti-interleukin-2 receptor agents such as basiliximab) (N = 253, 41.0%), or no induction (steroids) (N = 57, 9.2%). They studied reasonable numbers of patients and found no differences in outcomes such as rejection rates or renal function, reflected by serum creatinine at 1 y post–kidney after heart transplant. There was also no difference in recipient or graft survival. One difference that was noted was an increase in rehospitalization for the depletional induction group (51.7%) and nondepletional induction group (50.7%) versus the noninduction group (39.1%). The difference in rehospitalization rates between the noninduction group and the 2 induction groups while numerically lower did not reach statistical significance, likely due in part to the small numbers of patients in the noninduction group. Because the depletional group included patients receiving alemtuzumab, which is associated with profound lymphopenia, one wonders whether this exacerbated rehospitalization in this group.5 Details regarding the causes of rehospitalization were unfortunately not provided by the authors, but the presumption is that these would include lymphopenia and infection though neither appeared to impact survival or graft function. A reduction in rehospitalization is of considerable importance to reduce exposure of immunosuppressed patients to infections. There is also the issue of cost. While this article does not do a detailed cost analysis, it is quite clear that use of both depletional and nondepletional agents is far more expensive than the noninduction alternative, corticosteroids. When one adds the cost of additional rehospitalizations, the differential cost between the 2 induction strategies and the noninduction strategy would be excessive and ideally prohibitive. The authors sum up the preferred approach in their article: “No induction may be the preferred choice due to a lack of benefits and costly rehospitalizations associated with induction agents.”4 This statement should be tempered by the relatively small number of patients in the noninduction group and the lack of statistical differences as a result. There may well be patient groups such as sensitized heart transplant patients who might benefit from induction in kidney transplantation after heart transplant. Ideally, a randomized clinical trial of induction strategies versus noninduction would provide the most definitive information, but given the small numbers of patients who have kidney transplants after heart transplantation annually, this might take a long time to accomplish. It is important to remember the dictum Primum non Nocere: first, do no harm (either medical or financial).

Digitalizing heterologous gene expression in Gram‐negative bacteria with a portable ON/OFF module

While prokaryotic promoters controlled by signal‐responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription—as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation‐inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of, e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.

Identification of differentially regulated deceitful proteins in SH‐SY5Y cells engineered with Tet‐regulated protein expression system

Tetracycline regulated protein expression in mammalian cells is a powerful tool to predict the physiological function, cellular localization, and stability of a protein. In addition, to predict metabolic networks affected by the expression of wild-type or mutant forms of proteins, researchers generally produce a single mammalian cell clone that can express the protein of interest under tetracycline control and study the changes occurring in overall proteome before and after expression of a protein of interest. One limitation of tetracycline regulated clonal cell creation, however, is that it sometimes creates clones with changed protein levels even without the expression of the protein of interest due to the nonspecific insertion of the gene encoding the protein of interest into the genome or disruption of a metabolic pathway due to insertional silencing or activation. The aim of this study was to demonstrate the limitation of tetracycline regulated gene expression by creating clonal cell lines expressing the wild-type or the mutant forms of Fat mass and obesity-associated protein. Comparative proteome analysis of the protein extracts by two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF revealed the presence of eight proteins subjected to differential regulation even in the absence of induction. The identified proteins were 14-3-3 protein Epsilon, Vimentin, Heterogeneous nuclear ribonucleoprotein K, Tubulin beta-2C chain, Heat shock protein HSP 90-alpha, Heat shock protein HSP 90-beta, Alpha-enolase, TATA-binding protein-associated factor 2N. An ultimate care should be taken to prevent reporting of deceitful proteins generated from studies utilizing tetracycline regulated gene expression systems.

Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells

Death of cochlear hair cells, which do not regenerate, is a cause of hearing loss in a high percentage of the population. Currently, no approach exists to obtain large numbers of cochlear hair cells. Here, using asmall-molecule approach, we show significant expansion (>2,000-fold) of cochlear supporting cells expressing and maintaining Lgr5, an epithelial stem cell marker, in response to stimulation of Wnt signaling by a GSK3β inhibitor and transcriptional activation by a histone deacetylase inhibitor. The Lgr5-expressing cells differentiate into hair cells in high yield. From a single mouse cochlea, we obtained over 11,500 hair cells, compared to less than 200 in the absence of induction. The newly generated hair cells have bundles and molecular machinery for transduction, synapse formation, and specialized hair cell activity. Targeting supporting cells capable of proliferation and cochlear hair cell replacement could lead to the discovery of hearing loss treatments.

Fetal Growth and the Risk of Spontaneous Preterm Birth in a Prospective Cohort Study of Nulliparous Women.

Previous studies have suggested an association between fetal growth restriction and the risk of spontaneous preterm birth (sPTB). However, addressing this association is methodologically challenging. We conducted a prospective cohort study of nulliparous women with a singleton pregnancy in Cambridge, United Kingdom (2008-2012). Ultrasonic fetal biometry was performed at 20 weeks of gestation as per routine clinical care. Participants also had blinded research ultrasonography performed at approximately 28 weeks. Biometric measurements were expressed as gestational-age-adjusted z scores. Fetal growth velocity was quantified by change in z score between 20 weeks and 28 weeks. Risk of sPTB, defined as delivery at ≥28 weeks and <37 weeks associated with labor in the absence of induction, was analyzed using cause-specific Cox regression. Of 3,892 women, 98 (2.5%) had sPTB. When compared with the other decile groups, the lowest decile of growth velocity of the fetal femur between 20 and 28 weeks was associated with increased risk of sPTB (hazard ratio = 2.37, 95% confidence interval: 1.43, 3.93; P < 0.001). Adjustment for maternal characteristics had no material effect (hazard ratio = 2.50, 95% confidence interval: 1.50, 4.14; P < 0.001). There were no significant associations between other fetal measurements and risk of sPTB. To conclude, slow growth velocity of the fetal femur is associated with an increased risk of sPTB.

The Role of Transcription Factor Tfii-I/GTF2I in the Response to Cellular Stress in Hematopoietic Cells

The transcription factor ATF3 and the heme regulated eIF2α kinase (HRI) have previously been shown to be involved in the stress response in hematopoietic cells. Many stress-responsive genes are regulated at the level of transcription elongation and are characterized by paused RNA polymerase II (Pol II) at the promoter region. Indeed, analyzing data from the public ENCODE project revealed that the HRI and the ATF3 genes contained paused Pol II at the promoter and at upstream intergenic regulatory elements. In this study we performed a genomic and proteomic analysis of transcription factor TFII-I (also known as GTF2I) in human erythroleukemia K562 cells. TFII-I is a ubiquitously expressed transcription factor previously implicated in regulating genes involved in stress response. TFII-I exhibits both positive and negative effects on transcription. TFII-I is a relatively large protein (about 120 Kda) and contains DNA binding, nuclear localization, and multiple protein/protein interaction domains. Using biotinylation tagging technology and high-throughput sequencing, we determined sites of chromatin interactions for TFII-I. This study revealed that TFII-I binds downstream of Pol II peaks at the ATF3 and HRI gene loci (Fig 1). A proteomic analysis revealed that TFII-I interacts with the elongation factor elongin A, which has been shown to help Pol II overcome the paused state. We demonstrate that Elongin A only associates with the ATF3 gene in response to cellular stress. The stress induction of ATF3 gene transcription requires the function of TFII-I. Furthermore, in the absence of induction, transcription factors E2F6, and E2F4 as well as the histone deacetylase I (HDAC1) bind to the HRI and ATF3 genes in close proximity to the Pol II and TFII-I peaks (Fig. 1). E2F4/6 and HDAC1 have been implicated in the repression of gene expression. The DNA elements that recruit TFII-I, Pol II and the repressor proteins interact over long distances in K562 cells as shown by ChIA-Pet data available from the ENCODE project (Fig.1). Moreover, we demonstrate that TFII-I directly interacts with E2F6 using co-immunoprecipitation assays (Fig. 2). Partial depletion of TFII-I by shRNA led to a decrease in stress mediated transcription without impairing basal transcription levels. The data demonstrate that TFII-I regulates stress-responsive transcription of the HRI and ATF3 genes in hematopoietic cells. In the absence of stress, TFII-I occupies promoter and long distance regulatory elements together with Pol II, E2F6/E2F4, and HDAC1. These proteins may be part of a chromatin loop that keeps Pol II in a repressed configuration. We suggest that upon stress induction, TFII-I recruits Elongin A, which converts Pol II into a transcriptionally active configuration. Transcription elongation disrupts formation of a repressive chromatin loop and leads to transcription activation of the HRI and ATF3 genes. Figure 1 Diagram of the HRI and ATF3 gene loci showing interactions of TFII-I, Pol II, E2F4, E2F6, and HDAC1 as well as RNA seq, and Pol II ChIA-PET data. The ChIA-PET data show interactions of chromatin fragments that have Pol II bound. The arrows indicate the direction of transcription of the HRI gene and from the two promoters of the ATF3 gene (P1 and P2). Figure 1. Diagram of the HRI and ATF3 gene loci showing interactions of TFII-I, Pol II, E2F4, E2F6, and HDAC1 as well as RNA seq, and Pol II ChIA-PET data. The ChIA-PET data show interactions of chromatin fragments that have Pol II bound. The arrows indicate the direction of transcription of the HRI gene and from the two promoters of the ATF3 gene (P1 and P2). Figure 2 Input; TFII-I Figure 2. Input; TFII-I Figure 3 Co-Immunoprecipitation experiment showing the interaction between TFII-I and E2F6. K562 cell nuclear extracts were subjected to precipitation with IgG control or TFII-I specific antibodies. The material was fractionated by SDS-PAGE, transferred to a nylon membrane and probed with an antibody specific for E2F6. Figure 3. Co-Immunoprecipitation experiment showing the interaction between TFII-I and E2F6. K562 cell nuclear extracts were subjected to precipitation with IgG control or TFII-I specific antibodies. The material was fractionated by SDS-PAGE, transferred to a nylon membrane and probed with an antibody specific for E2F6. Disclosures No relevant conflicts of interest to declare.

Abstract 2817: DCVex(TM): A novel DC-targeted vector platform for cancer immunotherapy

Abstract Dendritic cells (DCs) are essential for the initiation of T cell responses and are therefore an attractive target for cancer immunotherapy. The DC vaccine, Provenge®, as well as a number of on-going clinical trials, have in principle validated this concept. However, the currently pursued strategy of ex vivo immunization of DCs is time-consuming and costly. We have developed a 3rd generation, integration-deficient lentivector platform, DCVex(TM), which is designed to deliver tumor antigen-encoding genes directly to DCs in vivo by targeting the DC-SIGN receptor. Mice immunized with DCVex(TM) vectors expressing a variety of model antigens developed strong, dose-dependent poly-functional and cytotoxic antigen-specific CD8 T cell responses, as assessed by intracellular cytokine staining and in vitro cytotoxic T lymphocyte assays. Repeated immunizations resulted in boosting of the T cell responses, indicating the absence of induction of inhibitory anti-vector immunity. Importantly, in stringent therapeutic tumor models (e.g., B16F10 footpad melanoma and CT26 lung metastasis models), immunization with tumor antigen-encoding vectors protected the majority of animals from death in a dose-dependent manner. These findings demonstrate the potential of DCVex(TM) as a novel cancer vaccine platform suitable for in vivo DC immunization. Citation Format: Tina C. Albershardt, Semih U. Tareen, Jared M. Odegard, David J. Campbell, Patrick Flynn, Scott H. Robbins, Peter Berglund, Jan H. ter Meulen. DCVex(TM): A novel DC-targeted vector platform for cancer immunotherapy. [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 2817. doi:10.1158/1538-7445.AM2014-2817

No evidence for induction or selection of mutant sodium channel expression in the copepod Acartia husdsonica challenged with the toxic dinoflagellate Alexandrium fundyense.

Some species in the dinoflagellate genus Alexandrium spp. produce a suite of neurotoxins that block sodium channels, known as paralytic shellfish toxins (PST), which have deleterious effects on grazers. Populations of the ubiquitous copepod grazer Acartia hudsonica that have co-occurred with toxic Alexandrium spp. are better adapted than naïve populations. The mechanism of adaptation is currently unknown. We hypothesized that a mutation in the sodium channel could account for the grazer adaptation. We tested two hypotheses: (1) Expression of the mutant sodium channel could be induced by exposure to toxic Alexandrium fundyense; (2) in the absence of induction, selection exerted by toxic A. fundyense would favor copepods that predominantly express the mutant isoform. In the copepod A. hudsonica, both isoforms are expressed in all individuals in varying proportions. Thus, in addition to comparing expression ratios of wild-type to mutant isoforms for individual copepods, we also partitioned copepods into three groups: those that predominantly express the mutant (PMI) isoform, the wild-type (PWI) isoform, or both isoforms approximately equally (EI). There were no differences in isoform expression between individuals that were fed toxic and nontoxic food after three and 6 days; induction of mutant isoform expression did not occur. Furthermore, the hypothesis that mutant isoform expression responds to toxic food was also rejected. That is, no consistent evidence showed that the wild-type to mutant isoform ratios decreased, or that the relative proportion of PMI individuals increased, due to the consumption of toxic food over four generations. However, in the selected line that was continuously exposed to toxic food sources, egg production rate increased, which suggested that adaptation occurred but was unrelated to sodium channel isoform expression.

Dose response and clonal variability of lentiviral tetracycline-regulated vectors in murine hematopoietic cells

Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells and provide the opportunity for time- and dose-controlled reversible transgene expression in hematopoietic stem cells in vitro and in vivo. However, the background activity of tetracycline-regulated promoters (tetPs) in the absence of induction or vector integration in the vicinity of proto-oncogenes can diminish the advantages of the system. Here we investigated the effect of lentiviral transduction rate on tetP background activity, vector copy number (VCN), and clonal variability as a consequence of vector integration. We found an exponential relationship between VCN and gene transfer/expression level, accompanied by a linear relationship between VCN and tetP background activity. Long-term murine transplantation studies demonstrated stable and reversible transgene expression in serial recipients. Although analysis of associated clonal composition revealed development of clonal dominance in the presence and absence of induction, no indications of disease presented during the observation period. The majority of tetracycline-regulated vector integration sites were identified in intron/exons of metabolic/housekeeping and signaling genes or in noncoding/repeat regions of the genome. Furthermore, we demonstrated that the nature of the selected transgene might affect tetP background activity and inducibility in vivo. Limiting tetP-regulated gene transfer may avoid generation of clones with high VCN and enhanced tetP background activity. Our data help to establish physiologic and pathophysiologic systems to study dose-dependent mechanisms triggered by different levels of transgene expression in the context of basic HSC biology and cellular transformation models.

Spheroid Formation and Enhanced Cardiomyogenic Potential of Adipose-Derived Stem Cells Grown on Chitosan

Mesenchymal stem cells may differentiate into cardiomyocytes and participate in local tissue repair after heart injury. In the current study, rat adipose-derived adult stem cells (ASCs) grown on chitosan membranes were observed to form cell spheroids after 3 days. The cell seeding density and surface modification of chitosan with Arg-Gly-Asp–containing peptide had an influence on the sizes of ASC spheroids. In the absence of induction, these spheroids showed an increased level of cardiac marker gene expression (Gata4, Nkx2-5, Myh6, and Tnnt2) more than 20-fold versus cells on the tissue culture polystyrene (TCPS) dish. Induction by 5-azacytidine or p38 MAP kinase inhibitor (SB202190) did not further increase the cardiac marker gene expression of these spheroids. Moreover, the enhanced cardiomyogenic potential of the spheroids was highly associated with the chitosan substrates. When ASC spheroids were plated onto TCPS with either basal or cardiac induction medium for 9 days, the spheroids spread into a monolayer and the positive effect on cardiomyogenic marker gene expression disappeared. The possible role of calcium ion and the up-regulation of adhesion molecule P-selectin and chemokine receptor Cxcr4 were demonstrated in ASC spheroids. Applying these spheroids to the chronic myocardial infarction animal model showed better functional recovery versus single cells after 12 weeks. Taken together, this study suggested that the ASC spheroids on chitosan may form as a result of calcium ion signaling, and the transplantation of these spheroids may offer a simple method to enhance the efficiency of stem cell–based therapy in myocardial infarction.

Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Gene therapy, in its current configuration, is irreversible and does not allow control over transgene expression in case of side effects. Only few regulated vector systems are available, and none of these has reached clinical applicability yet. The mifepristone (Mfp)-regulated Gene Switch (GS) system is characterized by promising features such as being composed of mainly human components and an approved small-molecule drug as an inducer. However, it has not yet been evaluated in adeno-associated virus (AAV) vectors, neither has it been tested for applicability in viral vectors in the central nervous system (CNS). Here, we demonstrate that the GS system can be used successfully in AAV vectors in the brain, and that short-term induced glial cell line-derived neurotrophic factor (GDNF) expression prevented neurodegeneration in a rodent model of Parkinson's disease (PD). We also demonstrate repeated responsiveness to the inducer Mfp and absence of immunological tissue reactions in the rat brain. Human equivalent dosages of Mfp used in this study were lower than those used safely for treatment of psychiatric threats, indicating that the inducer could be safely applied in patients. Our results suggest that the GS system in AAV vectors is well suited for further development towards clinical applicability.

Differences in Shiga toxin and phage production among stx2g-positive STEC strains

Shiga toxin-producing Escherichia coli (STEC) are characterized by the production of Shiga toxins (Stx) encoded by temperate bacteriophages. Stx production is linked to the induction of the phage lytic cycle. Several stx variants have been described and differentially associated with the risk of developing severe illness. The variant named stx2g was first identified in a STEC strain isolated from the faeces of healthy cattle. Analysis of stx2g-positive strains isolated from humans, animals, and environmental sources have shown that they have a close relationship. In this study, stx2g-positive STEC isolated from cattle were analyzed for phage and Stx production, with the aim to relate the results to differences observed in cytotoxicity. The presence of inducible phages was assessed by analyzing the bacterial growth/lysis curves and also by plaque assay. Bacterial growth curves in the absence of induction were similar for all isolates, however, notably differed among induced cultures. The two strains that clearly evidenced bacteriolysis under this condition also showed higher phage titers in plaque assays. However, only the phage plaques produced by one of these strains (FB 62) hybridized with a stx2-probe. Furthermore, the production of Stx was evaluated by enzyme immunoassay (EIA) and Western immunoblotting in overnight supernatants. By EIA, we detected Stx only in supernatants of FB 62, with a higher signal for induced than uninduced cultures. By immunoblotting, Stx2 could be detected after induction in all stx2g-positive isolates, but with lower amounts of Stx2B subunit in those supernatants where phages could not be detected. Taking into account all the results, several differences could be found among stx2g-positive strains. The strain with the highest cytotoxic titer showed higher levels of stx2-phages and toxin production by EIA, and the opposite was observed for strains that previously showed low cytotoxic titers, confirming that in stx2g-positive strains Stx production is phage-regulated.

Development of a Liver-specific Tet-On Inducible System for AAV Vectors and Its Application in the Treatment of Liver Cancer

Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet(bidir)Alb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet(bidir)CMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet(bidir)Alb was significantly higher than that of Tet(bidir)CMV, whereas leakage of Tet(bidir)Alb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet(bidir)-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet(bidir)-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.