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Genetic manipulation of hematopoietic stem and progenitor cells is an important tool for experimental and clinical applied hematology. However, techniques that allow for gene targeting, subsequent in vitro selection, and expansion of genetically defined clones are available only for ES cells. Such molecularly defined and, hence, \"safe\" clones would be highly desirable for somatic gene therapy. Here, we demonstrate that in vitro differentiated ES cells completely recapitulate the growth and differentiation properties of adult bone marrow cells, in vitro and in vivo, when ectopically expressing HOXB4. Myeloid development was enforced and (T) lymphoid development suppressed over a wide range of expression levels, whereas only high expression levels of the transcription factor were detrimental for erythroid development. This indicates a close association between the amounts of ectopic HOXB4 present within a progenitor cell and and the decision to self renew or differentiate. Because HOXB4 mediates similar fates of ES-derived and bone marrow hematopoietic stem cells, the primitive embryonic cells can be considered a promising alternative for investigating hematopoietic reconstitution, in vivo, based on well defined clones. Provided that HOXB4 levels are kept within a certain therapeutic window, ES cells also carry the potential of efficient and safe somatic gene therapy.

Homotypic vacuole fusion occurs by sequential priming, docking and fusion reactions. Priming frees the HOPS complex (Vps 11, 16, 18, 33, 39 and 41) to activate Ypt7p for docking. Here we explore the roles of the GDP and GTP states of Ypt7p using Gdi1p (which extracts Ypt7:GDP), Gyp7p (a GTPase‐activating protein for Ypt7p:GTP), GTPγS or GppNHp (non‐hydrolyzable nucleotides), and mutant forms of Ypt7p that favor either GTP or GDP states. GDP‐bound Ypt7p on isolated vacuoles can be extracted by Gdi1p, although only the GTP‐bound state allows docking. Ypt7p is converted to the GTP‐bound state after priming and stably associates with HOPS. Gyp7p can cause Ypt7p to hydrolyze bound GTP to GDP, driving HOPS release and accelerating Gdi1p‐mediated release of Ypt7p. Ypt7p extraction does not inhibit the Ca 2+ ‐triggered cascade that leads to fusion. However, in the absence of Ypt7p, fusion is still sensitive to GTPγS and GppNHp, indicating that there is a second specific GTPase that regulates the calcium flux and hence fusion. Thus, two GTPases sequentially govern vacuole docking and fusion.

Short-term, reversible expression of foreign proteins could be useful to modify cell fate. We have previously shown that retroviral vector mutants that are unable to initiate reverse transcription of their plus-stranded mRNA genomes mediate transient and highly efficient delivery of the site specific recombinase Cre into human and mouse fibroblasts (Galla et al., Mol Cell 2004). Cre protein activity was detected in >95% of target cells with a single treatment. Cre activity was particle-mediated and required the retroviral mRNA packaging signal as well as the expression of both Gag and Env in the packaging cell. Furthermore we could exclude plasmid contamination and Cre protein transduction as the underlying mechanisms for this mode of Cre delivery. Our data thus suggest that retrovirally delivered mRNA serves as an immediate translation template if not being reverse transcribed. When delivering the Sleeping Beauty (SB) transposase (cooperation with Z. Ivics, Berlin) by this approach, RT-deficient retroviral vector mutants were sufficient to mediate transposition of transfected plasmids containing transposon DNA. However, the efficiency of stable integration was only 3-fold above background levels, suggesting that the timing and/or expression levels have to be improved. Side-by-side comparison with the delivery of episomal DNA by lentiviral vectors revealed that RT-deficient retroviral mutants express foreign proteins for a much shorter duration (max. 4-5 days in replicating cells as opposed to >7 days for episomal lentiviral vectors) and lower levels (one to two orders of magnitude below the activity provided by episomal lentiviral vectors). As many receptor molecules require relatively low levels of expression to confer biological effects, we tested RT-deficient retroviral vectors to deliver the sialomucin CD34 or the murine cationic acid transporter mCAT-1, which serves as the receptor for the ecotropic murine leukemia virus. More than 80% of the exposed cells could be transduced following a single exposure to RT-deficient vectors encoding these receptors, and mCAT-1 expression conferred susceptibility of Jurkat cells to transduction with retroviral or lentiviral vectors pseudotyped with the murine ecotropic envelope protein. We conclude that RT-deficient retroviral vectors hold great promise for applications where low and transient expression of proteins achieves striking biological effects.

Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted pre-clinical model defined to assess their safety, in particular their risk related to insertional mutagenesis. In the murine pre-clinical study presented here, 40 test and 10 control mice were transplanted with ex vivo manipulated bone marrow cells to assess the long-term effects of the transduction of hematopoietic cells with the retroviral vector MSCV-MGMTP140Kwc. Test mice had significant gene marking 8–12 months post-transplantation with an average of 0.93 vector copies per cell and 41.5% of peripheral blood cells expressing the transgene MGMTP140K, thus confirming persistent vector expression. Unexpectedly, six test mice developed malignant lymphoma. No vector was detected in the tumor cells of five animals with malignancies, indicating that the malignancies were not caused by insertional mutagenesis or MGMTP140K expression. Mice from a concurrent study with a different transgene also revealed additional cases of vector-negative lymphomas of host origin. We conclude that the background tumor formation in this mouse model complicates safety determination of retroviral vectors and propose an improved study design that we predict will increase the relevance and accuracy of interpretation of pre-clinical mouse studies.

Severe adverse events related to insertional mutagenesis have reinforced interest in self-inactivating (SIN) retroviral vectors lacking enhancer-promoter sequences in the long terminal repeats (LTRs). Here, we have compared the potency of gammaretroviral and lentiviral vectors expressing the P140K mutant of O super(6)-methylguanine-DNA methyltransferase (MGMT). MGMT-P140K is a clinically relevant selection marker that mediates a strong survival advantage in hematopoietic cells exposed to alkylating agents. We designed gammaretroviral and lentiviral vectors that contained identical enhancer-promoter sequences located either in the LTR or downstream of the packaging region, for internal initiation of transcription from SIN backbones. Gammaretroviral vectors with intact LTRs containing enhancer-promoter sequences showed both higher titers and higher expression levels than the lentiviral counterparts, likely a result of suboptimal RNA processing of the lentiviral leader region. In the SIN context, gammaretroviral and lentiviral vectors with comparable internal cassettes had similar expression properties. Interestingly, gammaretroviral SIN vectors pseudotyped with RD114/TR had a higher transduction efficiency on proliferating human CD34 super(+) cells than lentiviral counterparts. These results encourage further investigations into the formation of retroviral hybrid vectors that combine the desired properties of high efficiency and increased biosafety.

Severe adverse events related to insertional mutagenesis have reinforced interest in self-inactivating (SIN) retroviral vectors lacking enhancer-promoter sequences in the long terminal repeats (LTRs). Here, we have compared the potency of gammaretroviral and lentiviral vectors expressing the P140K mutant of O(6)-methylguanine-DNA methyltransferase (MGMT). MGMT-P140K is a clinically relevant selection marker that mediates a strong survival advantage in hematopoietic cells exposed to alkylating agents. We designed gammaretroviral and lentiviral vectors that contained identical enhancer-promoter sequences located either in the LTR or downstream of the packaging region, for internal initiation of transcription from SIN backbones. Gammaretroviral vectors with intact LTRs containing enhancer-promoter sequences showed both higher titers and higher expression levels than the lentiviral counterparts, likely a result of suboptimal RNA processing of the lentiviral leader region. In the SIN context, gammaretroviral and lentiviral vectors with comparable internal cassettes had similar expression properties. Interestingly, gammaretroviral SIN vectors pseudotyped with RD114/TR had a higher transduction efficiency on proliferating human CD34(+) cells than lentiviral counterparts. These results encourage further investigations into the formation of retroviral hybrid vectors that combine the desired properties of high efficiency and increased biosafety.

Retroviral gene therapy vectors expressing the MGMTP140K transgene have been shown to protect hematopoietic cells from toxicity associated with a combined cancer treatment using 6-benzylguanine (6-BG) and an alkylating agent such as Temozolomide (TEM). In a Phase I gene transfer trial, high grade astrocytoma patients having poor prognoses using standard therapies, will undergo escalating dose treatments with 6-BG and TEM following MGMT P140K gene transfer into autologous hematopoietic stem cells. Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted preclinical model defined to assess their safety, and, in particular their risk related to insertional mutagenesis. This study was designed as a murine pre-clinical study to assess the long term effects of the transduction of hematopoietic cells with the retroviral vector to be used in the clinical trial, MSCV-MGMT P140K wc.LDBM cells from 5-FU treated C57BL/6 donors were transduced with ecotropic MSCV-MGMTP140K wc vector on recombinant fibronectin CH296 and transplanted into 40 lethally irradiated (11.75 Gy) C57BL/6 recipient test mice (10 controls received mock-transduced cells). Titer measured on non-hematopoietic cells led to an unintended high MOI on the transplanted cells of 4. The animals were observed for up to 12 months. Gene marking was determined by quantitative PCR and by intracellular staining of the human MGMT transgene product.All mice were found to have significant gene marking in the peripheral blood with 0.1-2 vector copies per cell. The majority of the animals (80%) demonstrated more than 40% peripheral blood cells expressing human MGMT protein 8-12 months post transplantation, thus confirming persistent vector expression. Unexpectedly, 5 test mice have been diagnosed with malignant lymphoma. None of the control mice have been found to have developed malignancies, although the full pathologic evaluation is pending for 5 of the 8 remaining control animals. Laser capture microdissection (LCM) of tumor cells with subsequent quantitative PCR detected no vector in the tumor cells of any of the 5 animals with malignancies, whereas vector was consistently detected in non-malignant hematopoietic tissue.These results indicate that the malignancies were not caused by insertional mutagenesis or MGMT P140K expression. Low numbers of control animals may explain the failure to observe malignancies in this group; however, further studies are required to exclude MSCV-MGMTP140K wc gene transfer as a causative factor for development of malignancies. A new murine study is initiated to distinguish host vs. donor cells, use a lower irradiation dose, include equal numbers of control animals, avoid 5-FU and utilize a transduction protocol with a MOI similar to the clinical protocol.

The COVID-19 pandemic has exposed an interconnected and tightly coupled globalized world in rapid change. This article sets the scientific stage for understanding and responding to such change for global sustainability and resilient societies. We provide a systemic overview of the current situation where people and nature are dynamically intertwined and embedded in the biosphere, placing shocks and extreme events as part of this dynamic; humanity has become the major force in shaping the future of the Earth system as a whole; and the scale and pace of the human dimension have caused climate change, rapid loss of biodiversity, growing inequalities, and loss of resilience to deal with uncertainty and surprise. Taken together, human actions are challenging the biosphere foundation for a prosperous development of civilizations. The Anthropocene reality— of rising system-wide turbulence—calls for transformative change towards sustainable futures. Emerging technologies, social innovations, broader shifts in cultural repertoires, as well as a diverse portfolio of active stewardship of human actions in support of a resilient biosphere are highlighted as essential parts of such transformations.

The aim of this study was to develop adequate in vitro conditions for the differentiation of bovine skeletal muscle cells. Therefore, satellite cells isolated from the left foreleg of a Holstein-Friesian fetus at 4.5 mo of gestation were seeded on 24-well plates coated with extracellular matrix gel. Cells were cultured for 5 d in growth medium containing 10% fetal bovine serum. After reaching confluence, several differentiation media were tested for inducing myotube formation. The highest fusion rate of approximately 30% was achieved with a serum-free medium containing 1 μM dexamethasone, 1 μg/ml linoleic acid, and 0.1 μM insulin after a differentiation phase of 72 h. Two different culture conditions (serum-free and serum-containing) appropriate for bovine skeletal muscle cell differentiation are described in detail which allow the investigation of bovine skeletal muscle cell proliferation and differentiation in general as well as in response to bioactive compounds.

Background The regulatory functions of microRNAs (miRNAs) in anti‐tumour immunity have been mainly described in immune effector cells. Since little is known about miRNA effects on the susceptibility of target cells during T cell—target cell interaction, this study focused on the identification of miRNAs expressed in tumour cells controlling their susceptibility to CD8+ T cell‐mediated cytotoxicity. Methods Luciferase expressing B16F10 melanoma (B16F10 Luci+) cells transfected with individual miRNAs covering a comprehensive murine miRNA library were screened for their susceptibility to lysis by an established cytotoxic T lymphocyte (CTL) line (5a, clone Nβ) specific for the melanoma‐associated antigen tyrosinase‐related protein 2. miRNAs with the most pronounced effects on T cell‐mediated lysis were validated and stably expressed in B16F10 cells. In silico analyses identified common targets of miRNA sets determined by the screen, which were further confirmed by small interfering RNA (siRNA)‐mediated silencing experiments modulating immune surveillance. The Ingenuity Pathway Analysis (IPA) software and RNA sequencing (RNA‐seq) data from miRNA‐overexpressing cell lines were applied to investigate the underlying mechanisms. The Cancer Genome Atlas (TCGA)‐derived miRNA sequencing data were used to assess the correlation of miRNA expression with melanoma patients’ survival. Results The miRNA screen resulted in the selection of seven miRNAs enhancing CTL‐mediated melanoma cell killing in vitro. Upon stable overexpression of selected miRNAs, hsa‐miR‐320a‐3p, mmu‐miR‐7037‐5p and mmu‐miR‐666‐3p were determined as most effective in enhancing susceptibility to CTL lysis. In silico analyses and subsequent siRNA‐mediated silencing experiments identified Psmc3 and Ndufa1 as common miRNA targets possibly involved in the functional effects observed. The analyses of RNA‐seq data with IPA showed pathways, networks, biological functions and key molecules potentially involved in the miRNA‐mediated functional effects. Finally, based on TCGA data analysis, a positive correlation of the conserved miRNAs among the panel of the seven identified miRNAs with overall survival of melanoma patients was determined. Conclusions For the first time, this study uncovered miRNA species that affect the susceptibility of melanoma cells to T cell‐mediated killing. These miRNAs might represent attractive candidates for novel therapy approaches against melanoma and other tumour entities. –miRNAs that significantly increase susceptibility of melanoma cells to CTL lysis were discovered. –RNA‐seq analyses show networks and pathways potentially involved in enhanced susceptibility to CTLs. –TCGA data analyses reveled correlations between expression of certain newly identified miRNAs and patient survival. –These miRNAs might promote the development of novel cancer therapies.