CREB is a critical regulator of normal hematopoiesis and leukemogenesis

The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation

Targeting lentiviral vectors to antigen-specific immunoglobulins

Gene transfer into B cells by lentivectors can provide an alternative approach to managing B lymphocyte malignancies and autoreactive B cell-mediated autoimmune diseases. These pathogenic B cell Populations can be distinguished by their surface expression of monospecific immunoglobulin. Development of a novel vector system to deliver genes to these specific B cells could improve the safety and efficacy of gene therapy. We have developed an efficient rnethod to target lentivectors to monospecific immunoglobulin-expressing cells in vitro and hi vivo. We were able to incorporate a model antigen CD20 and a fusogenic protein derived from the Sindbis virus as two distinct molecules into the lentiviral Surface. This engineered vector could specifically bind to cells expressing Surface immunoglobulin recognizing CD20 (αCD20), resulting in efficient transduction of target cells in a cognate antigen-dependent manner in vitro, and in vivo in a xenografted tumor model. Tumor suppression was observed in vivo, using the engineered lentivector to deliver a suicide gene to a xenografted tumor expressing αCD20. These results show the feasibility of engineering lentivectors to target immunoglobulin-specific cells to deliver a therapeutic effect. Such targeting lentivectors also Could potentially be used to genetically mark antigen-specific B cells in vivo to study their B cell biology

Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation

Figure S1. Feasibility of cryopreservation. (A) Tricistronic IDLV encoding for hGM-CSF, hIFN-α and CMV-pp65 protein used to generate SmyleDCpp65. (B) Scheme of SmyleDCpp65 generation. Monocytes were isolated by MACS selection, pre-conditioned with cytokines for 8 h, and transduced with IDLV-G2α2pp65 for 16 h. After transduction, cells were harvested and cryopreserved at 2x106 cells/mL/vial. Cells were analyzed immediately after thaw (AT) or cultured in medium without exogenous cytokines for 7 days. (C) Viability (7AADneg) and identity (CD14 + expression level) of cell product (AT). (D) Total IDLV copy numbers detected by RT-q-PCR in the transduced cell groups AT and after 7 days in culture. (E) pp65 expression in SmyleDCpp65 (CD14neg, CD11cbright) after 7 days of in vitro culture. (F) Viability, down regulation of monocyte marker (CD14), identity (CD11cbright and HLA-DR) and functional markers (CD86 and CD80) expressed in SmyleDCpp65 7 days after in vitro culture

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways - but in opposite directions - suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer

Elevated frequencies of leukemic myeloid and plasmacytoid dendritic cells in acute myeloid leukemia with the FLT3 internal tandem duplication

Some 30% of acute myeloid leukemia (AML) patients display an internal tandem duplication (ITD) mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3-ITDs are known to drive hematopoietic stem cells towards FLT3 ligand independent growth, but the effects on dendritic cell (DC) differentiation during leukemogenesis are not clear. We compared the frequency of cells with immunophenotype of myeloid DC (mDC: Lin−, HLA-DR+, CD11c+, CD86+) and plasmacytoid DC (pDC: Lin−, HLA-DR+, CD123+, CD86+) in diagnostic samples of 47 FLT3-ITD− and 40 FLT3-ITD+ AML patients. The majority of ITD+ AML samples showed high frequencies of mDCs or pDCs, with significantly decreased HLA-DR expression compared with DCs detectable in ITD− AML samples. Interestingly, mDCs and pDCs sorted out from ITD+ AML samples contained the ITD insert revealing their leukemic origin and, upon ex vivo culture with cytokines, they acquired DC morphology. Notably, mDC/pDCs were detectable concurrently with single lineage mDCs and pDCs in all ITD+ AML (n = 11) and ITD− AML (n = 12) samples analyzed for mixed lineage DCs (Lin−, HLA-DR+, CD11c+, CD123+). ITD+ AML mDCs/pDCs could be only partially activated with CD40L and CpG for production of IFN-α, TNF-α, and IL-1α, which may affect the anti-leukemia immune surveillance in the course of disease progression

Different modes and potencies of translational repression by sequence-specific RNA–protein interaction at the 5′-UTR

To determine whether sequence-specific RNA–protein interaction at the 5′-untranslated region (5′-UTR) can potently repress translation in mammalian cells, a bicistronic translational repression assay was developed to permit direct assessment of RNA–protein interaction and translational repression in transiently transfected living mammalian cells. Changes in cap-dependent yellow fluorescent protein (YFP) and internal ribosome entry sequence (IRES)-dependent cyan fluorescent protein (CFP) translation were monitored by fluorescence microscopy. Selective repression of YFP or coordinate repression of both YFP and CFP translation occurred, indicating two distinct modes by which RNA-binding proteins repress translation through the 5′-UTR. Interestingly, a single-stranded RNA-binding protein from Bacillus subtilis, tryptophan RNA-binding attenuation protein (TRAP), showed potent translational repression, dependent on the level of TRAP expression and position of its cognate binding site within the bicistronic reporter transcript. As the first of its class to be examined in mammalian cells, its potency in repression of translation through the 5′-UTR may be a general feature for this class of single-stranded RNA-binding proteins. Finally, a one-hybrid screen based on translational repression through the 5′-UTR identified linkers supporting full-translational repression as well as a range of partial repression by TRAP within the context of a fusion protein

Specific Inhibition of SRC Kinase Impairs Malignant Glioma Growth In Vitro and In Vivo

Malignant glioma is a severe cancer with a poor prognosis. Local occurrence and rare metastases of malignant glioma make it a suitable target for gene therapy. Several studies have demonstrated the importance of Src kinase in different cancers. However, these studies have focused mainly on Src-deficient mice or pharmacological inhibitors of Src. In this study we have used Src small hairpin RNAs (shRNAs) in a lentiviral backbone to mimic a long-term stable treatment and determined the role of Src in tumor tissues. Efficacy of Src shRNAs was confirmed in vitro demonstrating up to 90% target gene inhibition. In a mouse malignant glioma model, Src shRNA tumors were almost 50-fold smaller in comparison to control tumors and had significantly reduced vascularity. In a syngenic rat intracranial glioma model, Src shRNA-transduced tumors were smaller and these rats had a survival benefit over the control rats. In vivo treatment was enhanced by chemotherapy and histone deacetylase inhibition. Our results emphasise the importance of Src in tumorigenesis and demonstrate that it can be efficiently inhibited in vitro and in vivo in two independent malignant glioma models. In conclusion, Src is a potential target for RNA interference-mediated treatment of malignant glioma

In vivo bioluminescence imaging of locally disseminated colon carcinoma in rats

Animal tumour models using orthotopic tumours for the evaluation of cancer therapies are of greater clinical relevance than subcutaneous models, but they also pose greater difficulties for measuring tumour size and quantifying response to treatment. In this study, we used noninvasive bioluminescence imaging to monitor the intraperitoneal growth of luciferase-transfected CC531 colorectal cells in adult WAG/RIJ rats. The bioluminescence signal correlated well with post-mortem assessment of tumour load by visual inspection of the peritoneal cavity at specific follow-up times. Using bioluminescence imaging, we were able to monitor peritoneal tumour growth sequentially in time and to calculate a tumour growth rate for each animal; this is not possible with invasive methods of evaluating tumour load. Bioluminescence imaging of rats treated with a single dose of cisplatin (4 mg x kg(-1), i.p.) demonstrated a significant delay in peritoneal tumour growth relative to saline controls (mean 45.0+/-s.d. 13.0 vs 28.2+/-10.3 days; P=0.04). Similar protocols evaluated by visual scoring of tumour load at 40 days after inoculation supported these findings, although no quantitative assessment of treatment-induced growth delay could be made by this method. This study shows that in vivo imaging of luciferase-transfected tumour cells is a useful tool to investigate the dynamics of disseminated tumour growth and efficacy of anticancer treatment in orthotopic models of peritoneal cancer in rats. It offers an attractive alternative to invasive methods, and requires fewer animals for measuring tumour response to therapy

Innovations, challenges, and minimal information for standardization of humanized mice.

Mice xenotransplanted with human cells and/or expressing human gene products (also known as "humanized mice") recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human-specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community-driven resource called "Minimal Information for Standardization of Humanized Mice" (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice