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2.
Synth Biol (Oxf) ; 7(1): ysac026, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36452067

RESUMEN

Complex therapeutic antibody formats, such as bispecifics (bsAbs) or cytokine fusions, may provide new treatment options in diverse disease areas. However, the manufacturing yield of these complex antibody formats in Chinese Hamster Ovary (CHO) cells is lower than monoclonal antibodies due to challenges in expression levels and potential formation of side products. To overcome these limitations, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based knockout (KO) arrayed screening of 187 target genes in two CHO clones expressing two different complex antibody formats in a production-mimicking set-up. Our findings revealed that Myc depletion drastically increased product expression (>40%) by enhancing cell-specific productivity. The Myc-depleted cells displayed decreased cell densities together with substantially higher product titers in industrially-relevant bioprocesses using ambr15 and ambr250 bioreactors. Similar effects were observed across multiple different clones, each expressing a distinct complex antibody format. Our findings reinforce the mutually exclusive relationship between growth and production phenotypes and provide a targeted cell engineering approach to impact productivity without impairing product quality. We anticipate that CRISPR/Cas9-based CHO host cell engineering will transform our ability to increase manufacturing yield of high-value complex biotherapeutics.

4.
Cancers (Basel) ; 14(5)2022 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-35267468

RESUMEN

Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) was considered a promising treatment for patients with peritoneal metastasis from colorectal cancer. However, the recently published randomized controlled PRODIGE 7 trial failed to demonstrate survival benefits through the addition of short-term oxaliplatin-based HIPEC. Constituting a complex multifactorial treatment, we investigated HIPEC in a preclinical model concerning the elimination of minimal tumor residues, thereby aiming to better understand the size of effects and respective clinical trial results. Patient samples of peritoneal perfusates obtained during HIPEC treatments and oxaliplatin-containing solutions at clinically relevant dosages, conforming with established HIPEC protocols, were assessed regarding their ability to eliminate modelled ~100 µm thickness cancer cell layers. Impedance-based real-time cell analysis and classical end-point assays were used. Flow cytometry was employed to determine the effect of different HIPEC drug solvents on tumor cell properties. Effectiveness of peritoneal perfusate patient samples and defined oxaliplatin-containing solutions proved limited but reproducible. HIPEC simulations for 30 min reduced the normalized cell index below 50% with peritoneal perfusates from merely 3 out of 9 patients within 72 h, indicating full-thickness cytotoxic effects. Instead, prolonging HIPEC to 1 h enhanced these effects and comprised 7 patients' samples, while continuous drug exposure invariably resulted in complete cell death. Further, frequently used drug diluents caused approximately 25% cell size reduction within 30 min. Prolonging oxaliplatin exposure improved effectiveness of HIPEC to eliminate micrometastases in our preclinical model. Accordingly, insufficient penetration depth, short exposure time, and the physicochemical impact of drug solvents may constitute critical factors.

5.
Cell Stem Cell ; 28(5): 906-922.e6, 2021 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-33894142

RESUMEN

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can evolve to acute myeloid leukemia (AML). Mutations in CSF3R and RUNX1 are frequently observed in CN patients, although how they drive the transition from CN to AML (CN/AML) is unclear. Here we establish a model of stepwise leukemogenesis in CN/AML using CRISPR-Cas9 gene editing of CN patient-derived iPSCs. We identified BAALC upregulation and resultant phosphorylation of MK2a as a key leukemogenic event. BAALC deletion or treatment with CMPD1, a selective inhibitor of MK2a phosphorylation, blocked proliferation and induced differentiation of primary CN/AML blasts and CN/AML iPSC-derived hematopoietic stem and progenitor cells (HSPCs) without affecting healthy donor or CN iPSC-derived HSPCs. Beyond detailing a useful method for future investigation of stepwise leukemogenesis, this study suggests that targeting BAALC and/or MK2a phosphorylation may prevent leukemogenic transformation or eliminate AML blasts in CN/AML and RUNX1 mutant BAALC(hi) de novo AML.


Asunto(s)
Células Madre Pluripotentes Inducidas , Leucemia Mieloide Aguda , Proteínas de Neoplasias , Neutropenia , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Humanos , Leucemia Mieloide Aguda/genética , Mutación/genética , Proteínas de Neoplasias/genética , Neutropenia/congénito , Neutropenia/genética , Oncogenes
6.
Blood ; 134(14): 1159-1175, 2019 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-31366618

RESUMEN

Hematopoietic transcription factor LIM domain only 2 (LMO2), a member of the TAL1 transcriptional complex, plays an essential role during early hematopoiesis and is frequently activated in T-cell acute lymphoblastic leukemia (T-ALL) patients. Here, we demonstrate that LMO2 is activated by deacetylation on lysine 74 and 78 via the nicotinamide phosphoribosyltransferase (NAMPT)/sirtuin 2 (SIRT2) pathway. LMO2 deacetylation enables LMO2 to interact with LIM domain binding 1 and activate the TAL1 complex. NAMPT/SIRT2-mediated activation of LMO2 by deacetylation appears to be important for hematopoietic differentiation of induced pluripotent stem cells and blood formation in zebrafish embryos. In T-ALL, deacetylated LMO2 induces expression of TAL1 complex target genes HHEX and NKX3.1 as well as LMO2 autoregulation. Consistent with this, inhibition of NAMPT or SIRT2 suppressed the in vitro growth and in vivo engraftment of T-ALL cells via diminished LMO2 deacetylation. This new molecular mechanism may provide new therapeutic possibilities in T-ALL and may contribute to the development of new methods for in vitro generation of blood cells.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hematopoyesis , Proteínas con Dominio LIM/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Acetilación , Animales , Células Cultivadas , Células HEK293 , Humanos , Leucopoyesis , Ratones , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Pez Cebra
7.
Exp Hematol ; 71: 51-60, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30615903

RESUMEN

We describe the establishment of an embryoid-body-based protocol for hematopoietic/myeloid differentiation of human induced pluripotent stem cells that allows the generation of CD34+ cells or mature myeloid cells in vitro. Using this model, we were able to recapitulate the defective granulocytic differentiation in patients with severe congenital neutropenia (CN), an inherited preleukemia bone marrow failure syndrome. Importantly, in vitro maturation arrest of granulopoiesis was associated with an elevated unfolded protein response (UPR) and enhanced expression of the cell cycle inhibitor p21. Consistent with this, we found that CD34+ cells of CN patients were highly susceptible to DNA damage and showed diminished DNA repair. These observations suggest that targeting the UPR pathway or inhibiting DNA damage might protect hematopoietic cells of CN patients from leukemogenic transformation, at least to some extent.


Asunto(s)
Transformación Celular Neoplásica/metabolismo , Daño del ADN , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Leucemia/etiología , Modelos Biológicos , Neutropenia/congénito , Respuesta de Proteína Desplegada , Antígenos CD34/metabolismo , Biomarcadores , Células Cultivadas , Reprogramación Celular , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Estrés del Retículo Endoplásmico , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Células Madre Pluripotentes Inducidas/patología , Leucemia/metabolismo , Leucemia/patología , Neutropenia/etiología , Neutropenia/metabolismo , Neutropenia/patología
8.
Opt Express ; 20(5): 5481-500, 2012 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-22418354

RESUMEN

We introduce a 3-dimensional electromagnetic eigenmodal algorithm for the theoretical analysis of resonating nano-optical structures. The method, a variant of the Jacobi-Davidson algorithm, solves the electric field vector wave, or curl-curl, equation for the electromagnetic eigenmodes of resonant optical structures with a finite element method. In particular, the method includes transparent boundary conditions that enable the analysis of resonating structures in unbounded space. We demonstrate the performance of the method. First, we calculate the modes of several dielectric resonator antennas and compare them to theoretically determined results. Second, we calculate the modes of a nano-cuboid and compare them to theoretically determined results. Third, we numerically analyze spherical nanoparticles and compare the result to the theoretical Mie solution. Fourth, we analyze optical dipole antenna configurations in order to assess the method's capability for solving technologically relevant problems.


Asunto(s)
Modelos Químicos , Modelos Moleculares , Nanoestructuras/química , Nanoestructuras/ultraestructura , Resonancia por Plasmón de Superficie/métodos , Simulación por Computador , Luz , Dispersión de Radiación
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