Isoprenoids constitute one of the most diverse classes of natural products. As a compound class, they are essential to basic metabolic processes including cell-wall biosynthesis, post-translational ...protein modifications, and signaling. In addition, isoprenoid secondary metabolites are highly valuable natural products with a wide range of biotechnological applications. The biosynthesis of their two universal building blocks, isopentenyl diphosphate and dimethylallyl diphosphate, was thought to proceed exclusively by way of mevalonate as a key intermediate until a novel pathway involving methylerithritol phosphate (MEP) was discovered in the early 1990s. In this review, we describe the seven enzymes of the MEP pathway, along with their discoveries, three-dimensional structures, and mechanisms. The latter include examples of remarkable enzyme catalysis including an unusual cytidilation reaction and the use of iron–sulfur cluster cofactors in reductive ring opening and hydroxy-group elimination. Furthermore, isoprenoid biosynthesis shows a characteristic species distribution. A brief overview highlights the MEP pathway’s potential as a selective drug target, which is absent in humans but essential to the survival of many important bacterial and apicomplexan pathogens.
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IJS, KILJ, NUK, PNG, UL, UM
T cells modified with CD19-specific chimeric antigen receptors (CARs) result in significant clinical benefit for leukemia patients but constitute a challenge for manufacturing. We have recently ...demonstrated the in vivo generation of CD19-CAR T cells using the CD8-targeted lentiviral vector (CD8-LV). In this study, we investigated the in vivo generation of CD4+ CAR T cells using CD4-targeted LV (CD4-LV). Administration of CD4-LV into NSG mice transplanted with human peripheral blood mononuclear cells (PBMCs) led to 40%–60% of human CD4+ lymphocytes being CAR positive while CD8+ cells remained CAR negative. CAR+ T cells displayed a T helper 1 (Th1)/Th2 phenotype, which was accompanied by CD19+ B cell elimination. Intravenous administration of CD4-LV into NSG mice reconstituted with human CD34+ cells induced CAR expression and B cell elimination within 2–3 weeks post-injection. Preclinical analysis in a tumor mouse model revealed that mice administered CD4-LV exhibited faster and superior tumor cell killing compared to mice injected with CD8-LV alone or as a mixture with CD4-LV. Further analysis suggests that CD4+CAR+ cells may outperform CD8+CAR+ cells, especially at a high burden of target antigen, mainly since CD8 cells are more prone to exhaustion. This first description of in vivo-generated CD4+ CAR T cells supports their importance for cellular therapy.
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In clinics, CAR T cell products are comprised of both CD4+ and CD8+ T cells, but CD8+ T cells are preferred due to their cytolytic activities. This study outlines the importance of CD4 T cells for in vivo-generated CAR T cells using CD4-targeted lentiviral vectors (CD4-LVs). Mice administered CD4-LV displayed faster kinetics and were superior in killing tumor cells compared to mice administered CD8-LV or a mixture of the two vectors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Chimeric antigen receptor (CAR) T cells brought substantial benefit to patients with B‐cell malignancies. Notwithstanding, CAR T‐cell manufacturing requires complex procedures impeding the broad ...supply chain. Here, we provide evidence that human CD19‐CAR T cells can be generated directly in vivo using the lentiviral vector CD8‐LV specifically targeting human CD8+ cells. Administration into mice xenografted with Raji lymphoma cells and human peripheral blood mononuclear cells led to CAR expression solely in CD8+ T cells and efficacious elimination of CD19+ B cells. Further, upon injection of CD8‐LV into mice transplanted with human CD34+ cells, induction of CAR T cells and CD19+ B‐cell depletion was observed in 7 out of 10 treated animals. Notably, three mice showed elevated levels of human cytokines in plasma. Tissue‐invading CAR T cells and complete elimination of the B‐lymphocyte‐rich zones in spleen were indicative of a cytokine release syndrome. Our data demonstrate the feasibility of in vivo reprogramming of human CD8+ CAR T cells active against CD19+ cells, yet with similar adverse effects currently notorious in the clinical practice.
Synopsis
T cells were reprogrammed with chimeric antigen receptors (CARs) in situ upon systemic injection of CD8‐targeted lentiviral vectors, thus providing proof‐of‐principle for converting CAR T cell therapy from an autologous, individualized to an off‐the‐shelf product.
In situ CAR T cell generation was evidenced in mice transplanted with human PBMC or CD34+ stem cells.
In situ generated CAR T cells expanded upon antigen recognition and eliminated CD19+ cells.
Some mice developed side‐effects resembling the cytokine release syndrome observed in patients treated with CAR T cells.
T cells were reprogrammed with chimeric antigen receptors (CARs) in situ upon systemic injection of CD8‐targeted lentiviral vectors, thus providing proof‐of‐principle for converting CAR T cell therapy from an autologous, individualized to an off‐the‐shelf product.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Lymphocytes have always been among the prime targets in gene therapy, even more so since chimeric antigen receptor (CAR) T cells have reached the clinic. However, other gene therapeutic approaches ...hold great promise as well. The first part of this review provides an overview of current strategies in lymphocyte gene therapy. The second part highlights the importance of precise gene delivery into B and T cells as well as distinct subtypes of lymphocytes. This can be achieved with lentiviral vectors (LVs) pseudotyped with engineered glycoproteins recognizing lymphocyte surface markers as entry receptors. Different strategies for envelope glycoprotein engineering and selection of the targeting ligand are discussed. With a CD8-targeted LV that was recently used to achieve proof of principle for the
reprogramming of CAR T cells, these vectors are becoming a key tool to genetically engineer lymphocytes directly
.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The development of cost-effective and sustainable catalytic methods for the production of enantiomerically pure chiral amines is a key challenge facing the pharmaceutical and fine chemical ...industries. This challenge is highlighted by the estimate that 40–45% of drug candidates contain a chiral amine, fueling a demand for broadly applicable synthetic methods that deliver target structures in high yield and enantiomeric excess. Herein we describe the development and application of a “toolbox” of monoamine oxidase variants from Aspergillus niger (MAO-N) which display remarkable substrate scope and tolerance for sterically demanding motifs, including a new variant, which exhibits high activity and enantioselectivity toward substrates containing the aminodiphenylmethane (benzhydrylamine) template. By combining rational structure-guided engineering with high-throughput screening, it has been possible to expand the substrate scope of MAO-N to accommodate amine substrates containing bulky aryl substituents. These engineered MAO-N biocatalysts have been applied in deracemization reactions for the efficient asymmetric synthesis of the generic active pharmaceutical ingredients Solifenacin and Levocetirizine as well as the natural products (R)-coniine, (R)-eleagnine, and (R)-leptaflorine. We also report a novel MAO-N mediated asymmetric oxidative Pictet–Spengler approach to the synthesis of (R)-harmicine.
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IJS, KILJ, NUK, PNG, UL, UM
Vanadium‐dependent haloperoxidases (VHPOs) are a class of halogenating enzymes found in fungi, lichen, algae, and bacteria. We report the cloning, purification, and characterization of a functional ...VHPO from the cyanobacterium Acaryochloris marina (AmVHPO), including its structure determination by X‐ray crystallography. Compared to other VHPOs, the AmVHPO features a unique set of disulfide bonds that stabilize the dodecameric assembly of the protein. Easy access by high‐yield recombinant expression, as well as resistance towards organic solvents and temperature, together with a distinct halogenation reactivity, make this enzyme a promising starting point for the development of biocatalytic transformations.
A handy haloperoxidase: The structural and functional characterization of a cyanobacterial vanadium‐dependent haloperoxidase is reported. Easy access by recombinant expression, robustness to organic reaction conditions, and a distinct bromination reactivity make this enzyme a promising tool for biocatalytic halogenations.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Chimeric antigen receptor (CAR)-expressing T cells are a complex and heterogeneous gene therapy product with variable phenotype compositions. A higher proportion of less differentiated CAR T cells is ...usually associated with improved antitumoral function and persistence. We describe in this study a novel receptor-targeted lentiviral vector (LV) named 62L-LV that preferentially transduces less differentiated T cells marked by the L-selectin receptor CD62L, with transduction rates of up to 70% of CD4+ and 50% of CD8+ primary T cells. Remarkably, higher amounts of less differentiated T cells are transduced and preserved upon long-term cultivation using 62L-LV compared to VSV-LV. Interestingly, shed CD62L neither altered the binding of 62L-LV particles to T cells nor impacted their transduction. The incubation of 2 days of activated T lymphocytes with 62L-LV or VSV-LV for only 24 hours was sufficient to generate CAR T cells that controlled tumor growth in a leukemia tumor mouse model. The data proved that potent CAR T cells can be generated by short-term
ex vivo
exposure of primary cells to LVs. As a first vector type that preferentially transduces less differentiated T lymphocytes, 62L-LV has the potential to circumvent cumbersome selections of T cell subtypes and offers substantial shortening of the CAR T cell manufacturing process.
Autism spectrum disorder (ASD) is a complex heterogeneous neurodevelopmental disorder characterized by social and communicative impairments, as well as repetitive and restricted behaviors (RRBs). ...With the limited effectiveness of current pharmacotherapies in treating repetitive behaviors, the present study determined the effects of acute systemic treatment of the novel multi-targeting ligand ST-2223, with incorporated histamine H3 receptor (H3R) and dopamine D2/D3 receptor affinity properties, on ASD-related RRBs in a male Black and Tan BRachyury (BTBR) mouse model of ASD. ST-2223 (2.5, 5, and 10 mg/kg, i.p.) significantly mitigated the increase in marble burying and self-grooming, and improved reduced spontaneous alternation in BTBR mice (all p < 0.05). Similarly, reference drugs memantine (MEM, 5 mg/kg, i.p.) and aripiprazole (ARP, 1 mg/kg, i.p.), reversed abnormally high levels of several RRBs in BTBR (p < 0.05). Moreover, ST-2223 palliated the disturbed anxiety levels observed in an open field test (all p < 0.05), but did not restore the hyperactivity parameters, whereas MEM failed to restore mouse anxiety and hyperactivity. In addition, ST-2223 (5 mg/kg, i.p.) mitigated oxidative stress status by decreasing the elevated levels of malondialdehyde (MDA), and increasing the levels of decreased glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) in different brain parts of treated BTBR mice (all p < 0.05). These preliminary in vivo findings demonstrate the ameliorative effects of ST-2223 on RRBs in a mouse model of ASD, suggesting its pharmacological prospective to rescue core ASD-related behaviors. Further confirmatory investigations on its effects on various brain neurotransmitters, e.g., dopamine and histamine, in different brain regions are still warranted to corroborate and expand these initial data.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
In vitro cofactor supply and regeneration have been a major obstacle for biocatalytic processes, in particular on a large scale. Peroxidases often suffer from inactivation by their oxidative ...co‐factor. Combining photocatalysis and biocatalysis offers an innovative solution to this problem, but lacks atom economy due to the sacrificial electron donors needed. Herein, we show that redox‐active buffers or even water alone can serve as efficient, biocompatible electron sources, when combined with photocatalysis. Mechanistic investigations revealed first insights into the possibilities and limitations of this approach and allowed adjusting the reaction conditions to the specific needs of biocatalytic transformations. Proof‐of‐concept for the applicability of this photobiocatalytic reaction setup was given by enzymatic halogenations.
Photo meets bio: Photobiocatalysis constitutes an elegant way to overcome the pending problem of cofactor inactivation in peroxidase catalyzed transformations. In this context, the utility of redox‐active buffers or even water alone as efficient, biocompatible electron sources has been demonstrated by combining photocatalytic in situ H2O2 generation with enzymatic halogenations.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
CD3-targeted lentiviral vectors (CD3-LVs) mediate selective transduction of human T lymphocytes in vitro and in vivo while simultaneously activating the targeted cells. Previously, we have ...demonstrated that CD3-LV leads to downmodulation of the CD3:T cell receptor (TCR) complex. We therefore hypothesized that inhibition of CD3 phosphorylation by Src/Abl tyrosine kinase inhibitors such as dasatinib results in enhancement of gene delivery by T cell-targeted LVs. Indeed, dasatinib treatment of T cells prior to incubation with CD3-LV increased reporter gene delivery by 3- to 10-fold. Moreover, the presence of dasatinib enhanced selective transduction into non-activated target cells present in whole blood. When combined with delivery of the CD19-chimeric antigen receptor (CAR) gene, dasatinib increased CAR T cell numbers by close to 10-fold. Importantly, the short-term exposure of T cells to dasatinib during vector incubation did not interfere with tumor cell killing by the resulting CAR T cells and rather came along with less upregulated exhaustion markers and a more naive phenotype. Our data suggest that dasatinib prevents CD3-LV-induced phosphorylation and CD3:TCR intake, thereby increasing the amount of CD3-LV bound to the cell surface. This is the first description of dasatinib as transduction enhancer, an activity particularly relevant for CAR T cell generation with CD3-LV.
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This paper describes the discovery of dasatinib as transduction enhancer for gene delivery into T lymphocytes by CD3-targeted lentiviral vectors. Dasatinib acts through preventing CD3:TCR intake, thereby increasing the amount of vector particles bound to the T cell surface. Improved generation of CAR T cells is illustrated as an immediate application.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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