CRISPR/Cas9 mediated knockout of microRNAs for precise cell engineering

Recent advances in the development of molecular tools available for cell line engineering has opened avenues for precise modulation of production cell lines to optimize cellular process relevant parameters. Among those, the CRISPR/Cas9 system represents a rapid and straightforward cell line engineering tool which allows for precise gene editing of host genomes to modify relevant signaling pathways. In addition, microRNAs (miRNAs) have proven to serve as versatile molecular tools to improve production cells regarding protein production and growth characteristics. These small non-coding RNA molecules are regulators of gene expression and regulate process relevant cellular pathways as growth, apoptosis, protein expression or stress. With an individual microRNA being able to regulate up to several hundred target genes, the modulation of miRNA expression may mimic simultaneous modification of several hundred target genes. Therefore, miRNAs may offer great potential to serve as targets for CRISPR/Cas9-mediated genome editing, as an individual microRNA can regulate up to several hundred target genes and therefore mimics multiplexing of hundreds of target genes in one single step. While the overexpression of miRNAs to improve the performance of biopharmaceutical production hosts has received major interest in the past years, effects of precise knockout of unfavorable miRNAs in Chinese hamster ovary (CHO) production cells have not been reported yet. Therefore, the current study aimed at applying a novel strategy to increase product yields by enhancing viability and culture longevity of CHO cells using CRISPR/Cas9-mediated deletion of a miRNA causing adverse effects. In a previous high content screen with more than 1000 miRNAs we were able to identify miR-744 as being involved in the regulation of apoptosis. After the apoptosis-inducing capacity of miR-744 in monoclonal antibody-producing CHO-mAb1 cells was revalidated, the genomic precursor sequence of miR-744 was deleted by two sgRNA-Cas9-mediated DNA double-strand breaks flanking the miR-744 gene in the CHO genome. A plasmid encoding both required sgRNAs in a tandem array, Cas9 and GFP was used to allow for simultaneous delivery of all required components and thereby reducing the screening effort for potential knockout (KO) cell lines. After fluorescent-activated cell sorting (FACS) seven putative, clonal miR-744-KO cell lines could be recovered. From these, three were identified and confirmed as miR-744-KOs by sequencing of their miR-744 locus respectively. In a subsequent growth experiment over a period of nine days considerable differences in the investigated parameters viable cell count, viability, apoptosis, necrosis and antibody titers of the miR-744-KO cell lines in comparison to untreated cells and non-targeting sgRNA transfected cells could be observed. Further experiments are conducted to analyze the observed effects. In summary, the present study elucidates the role of microRNAs, which bear adverse effects on production cells, as targets for the novel CRISPR/Cas9 genome editing technique in the context of cell line engineering. This approach contributes to the idea of a rational design of CHO production hosts with controlled modulation of signaling pathways or metabolic characteristics to enable the generation of optimized production cells

New Expression Method and Characterization of Recombinant Human Granulocyte Colony Stimulating Factor in a Stable Protein Formulation

Human recombinant granulocyte colony stimulating factor (rhG-CSF) is widely used in hematology and oncology for the treatment of neutropenia, for the restoration of neutrophil production after bone marrow transplantation, for myelodysplastic syndromes, and aplastic anemia. The E. coli expression system is commonly used for fast recombinant production of rhG-CSF at a large scale. We have applied a novel autoinduction method for the batch expression of rhG-CSF to study whether this new system would increase cell mass and target-protein yield compared to conventional E. coli cell culture and induction with isopropyl ?-D-thiogalactopyranoside (IPTG). We could demonstrate 3-fold higher culture densities and a 5-fold higher protein yield compared to IPTG induction without the need to monitor cell growth in a shortened 24 h expression procedure. rhG-CSF expressed in autoinduction media was successfully extracted from E. coli inclusion bodies and refolded by dialysis. After size exclusion chromatography (SEC) purification, rhG-CSF showed similar conformation, biological activity and aggregation profile compared to the commercially available biosimilar TEVAgrastim® (TEVA Pharma AG). Expression by autoinduction is suggested as a cost- and time-effective method for rhG-CSF production

The Akt-inhibitor Erufosine induces apoptotic cell death in prostate cancer cells and increases the short term effects of ionizing radiation

<p>Abstract</p> <p>Background and Purpose</p> <p>The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is frequently deregulated in prostate cancer and associated with neoplastic transformation, malignant progression, and enhanced resistance to classical chemotherapy and radiotherapy. Thus, it is a promising target for therapeutic intervention. In the present study, the cytotoxic action of the Akt inhibitor Erufosine (ErPC3) was analyzed in prostate cancer cells and compared to the cytotoxicity of the PI3K inhibitor LY294002. Moreover, the efficacy of combined treatment with Akt inhibitors and ionizing radiation in prostate cancer cells was examined.</p> <p>Materials and methods</p> <p>Prostate cancer cell lines PC3, DU145, and LNCaP were treated with ErPC3 (1-100 µM), LY294002 (25-100 µM), irradiated (0-10 Gy), or subjected to combined treatments. Cell viability was determined by the WST-1 assay. Apoptosis induction was analyzed by flow cytometry after staining with propidium iodide in a hypotonic citrate buffer, and by Western blotting using antibodies against caspase-3 and its substrate PARP. Akt activity and regulation of the expression of Bcl-2 family members and key downstream effectors involved in apoptosis regulation were examined by Western blot analysis.</p> <p>Results</p> <p>The Akt inhibitor ErPC3 exerted anti-neoplastic effects in prostate cancer cells, however with different potency. The anti-neoplastic action of ErPC3 was associated with reduced phosphoserine 473-Akt levels and induction of apoptosis. PC3 and LNCaP prostate cancer cells were also sensitive to treatment with the PI3K inhibitor LY294002. However, the ErPC3-sensitive PC3-cells were less susceptible to LY294002 than the ErPC3-refractory LNCaP cells. Although both cell lines were largely resistant to radiation-induced apoptosis, both cell lines showed higher levels of apoptotic cell death when ErPC3 was combined with radiotherapy.</p> <p>Conclusions</p> <p>Our data suggest that constitutive Akt activation and survival are controlled by different different molecular mechanisms in the two prostate cancer cell lines - one which is sensitive to the Akt-inhibitor ErPC3 and one which is more sensitive to the PI3K-inhibitor LY294002. Our findings underline the importance for the definition of predictive biomarkers that allow the selection patients that may benefit from the treatment with a specific signal transduction modifier.</p

A New Type of Thermoalkalophilic Hydrolase of Paucimonas lemoignei with High Specificity for Amorphous Polyesters of Short Chain-length Hydroxyalkanoic Acids

A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei. Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholate-coated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units. The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate). The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively. No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-alpha-benzoyl-l-arginine-4-nitranilide, or starch. The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g. n-propyl alcohol). The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents. Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases. A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J. L., and Jaeger, K.-E. (1999) Biochem. J. 343, 177-183) is suggested

Protein Kinase C Delta (PKCδ) Affects Proliferation of Insulin-Secreting Cells by Promoting Nuclear Extrusion of the Cell Cycle Inhibitor p21Cip1/WAF1

BACKGROUND:High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. METHODOLOGY AND PRINCIPAL FINDINGS:Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21(Cip1/WAF1). This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21(Cip1/WAF1) at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21(Cip1/WAF1) was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21(Cip1/WAF1) with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. CONCLUSIONS AND SIGNIFICANCE:These observations disclose PKCδ as negative regulator of p21(Cip1/WAF1), which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role

Proapoptotic activity of Ukrain is based on Chelidonium majus L. alkaloids and mediated via a mitochondrial death pathway

BACKGROUND: The anticancer drug Ukrain (NSC-631570) which has been specified by the manufacturer as semisynthetic derivative of the Chelidonium majus L. alkaloid chelidonine and the alkylans thiotepa was reported to exert selective cytotoxic effects on human tumour cell lines in vitro. Few clinical trials suggest beneficial effects in the treatment of human cancer. Aim of the present study was to elucidate the importance of apoptosis induction for the antineoplastic activity of Ukrain, to define the molecular mechanism of its cytotoxic effects and to identify its active constituents by mass spectrometry. METHODS: Apoptosis induction was analysed in a Jurkat T-lymphoma cell model by fluorescence microscopy (chromatin condensation and nuclear fragmentation), flow cytometry (cellular shrinkage, depolarisation of the mitochondrial membrane potential, caspase-activation) and Western blot analysis (caspase-activation). Composition of Ukrain was analysed by mass spectrometry and LC-MS coupling. RESULTS: Ukrain turned out to be a potent inducer of apoptosis. Mechanistic analyses revealed that Ukrain induced depolarisation of the mitochondrial membrane potential and activation of caspases. Lack of caspase-8, expression of cFLIP-L and resistance to death receptor ligand-induced apoptosis failed to inhibit Ukrain-induced apoptosis while lack of FADD caused a delay but not abrogation of Ukrain-induced apoptosis pointing to a death receptor independent signalling pathway. In contrast, the broad spectrum caspase-inhibitor zVAD-fmk blocked Ukrain-induced cell death. Moreover, over-expression of Bcl-2 or Bcl-x(L )and expression of dominant negative caspase-9 partially reduced Ukrain-induced apoptosis pointing to Bcl-2 controlled mitochondrial signalling events. However, mass spectrometric analysis of Ukrain failed to detect the suggested trimeric chelidonine thiophosphortriamide or putative dimeric or monomeric chelidonine thiophosphortriamide intermediates from chemical synthesis. Instead, the Chelidonium majus L. alkaloids chelidonine, sanguinarine, chelerythrine, protopine and allocryptopine were identified as major components of Ukrain. Apart from sanguinarine and chelerythrine, chelidonine turned out to be a potent inducer of apoptosis triggering cell death at concentrations of 0.001 mM, while protopine and allocryptopine were less effective. Similar to Ukrain, apoptosis signalling of chelidonine involved Bcl-2 controlled mitochondrial alterations and caspase-activation. CONCLUSION: The potent proapoptotic effects of Ukrain are not due to the suggested "Ukrain-molecule" but to the cytotoxic efficacy of Chelidonium majus L. alkaloids including chelidonine

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Novel poly(3-hydroxybutyrat) depolymerases of paucimonas lemoignei and rhodospirillum rubrum

Aus Paucimonas lemoignei-Kulturüberstand wurde eine neuartige unter Kohlenstoffmangel während des späten exponentiellen Wachstums auf organischen Säuren oder parakristallinen dPHASCL {denaturierte Poly[(R)-3-Hydroxyalkanoate]; Monomeruntereinheiten C3-C5} gebildete PHASCL Depolymerase (PhaZ7; 36.2 kDa, stabil bis pH 12, 60°C) isoliert. Das Enzym hydrolysierte amorphe nPHASCL (zu 3-HA Pentamer) und ataktische P[(R,S)-3-HB]. PHAMCL (> C6), Polynukleotide, Proteasesubstrate, Poly(6-Hydroxyhexanoat), Lipide wurden nicht hydrolysiert. phaZ7 mit SacB-Signalsequenz wurde heterolog in Bacillus subtilis expremiert. Partielle Homologien zu Bacillus Lipasen und ortsgerichte Mutagenese präferieren S136, D242, H306 (H47G48 Oxyanion) als katalytische Triade. PhaZ7 entspricht dem „dPHB Depolymerase Inhibitor“ (MUKAI et al., 1992). Substratspezifität/kinetischen Studien wiesen auf eine Kooperativität der PHASCL Depolymerasen aus P. lemoignei hin. Intrazelluläre nPHASCL Depolymerase aus Rhodospirillum rubrum (PhaZ1Rr) wurde gereinigt und charakterisiert. Die nPHB-Hydrolyse in vitro erforderte zwei lösliche Komponenten: thermostabilen „Aktivator“ und thermolabile Depolymerase (MERRICK & DOUDOROFF, 1964). Vorinkubation von nPHB mit Aktivator oder Proteasen erhöhte die Hydrolyserate (> 6x). Der Aktivator war Protease-sensitiv, stabil in Alkanen, Ketonen, Alkoholen und schwach polaren Lösungsmitteln. PhaZ1Rr (35kDa, Topt.50°C, pHopt.9) hydrolysierte nPHB (3.1mmol cuts min-1 mg-1) unabhängig von Granula-Ursprung und Art der Aktivierung zu 3-HA-Mono-, Di- und Trimer, besaß jedoch keine Lipase-, Protease- oder Esteraseaktivität (lösliche Ester). phaZ1Rr wurde in E. coli heterolog expremiert. Die Aminosäuresequenz war homolog zu dPHASCL-Depolymerasen Typ 2. Linker- und Substratbindedomäne fehlten. Die Lipasebox (GIS19SG) entsprach PHAMCL Depolymerasen. Sequenzhomologien und Sekundärstrukturanalysen weisen auf S19, D115, H155 (H250G251 Oxyanion) als katalytische Triade hin.First extracellular nPHASCL {amorphous poly[(R)-3-hydroxyalkanoic acid]} depolymerase (PhaZ7) was purified from Paucimonas lemoignei. The enzyme (36.2 kDa, stable < pH 12, 60°C) was secreted in late exponential growth phase as on paracrystalline dPHASCL in response to carbon starvation. It hydrolyzed nPHASCL releasing 3-HA pentamer. Shown on atactic poly[(R,S)-3-HB] PhaZ7 recognized the physical state of the polymer. PHAMCL, polynucleotides, protease substrates, poly(6-hydroxyhexanoate) and lipids were not hydrolyzed. phaZ7 with SacB signal sequence was functionally expressed in Bacillus subtilis. Only limited homologies to lipases (Bacillus) were found. Sequence homologies and mutagenesis suggest a catalytical triad of S136, D242, H306 (H47G48 oxyanion). PhaZ7 was „dPHB depolymerase inhibitor“ (MUKAI et al., 1992). Substrate specificity/kinetic studies suggest that seven PHASCL depolymerases by P. lemoignei could provide a cooperative effect. Intracellular nPHASCL depolymerase from Rhodospirillum rubrum (PhaZ1Rr) was purified and characterized. Two soluble components were necessary for hydrolysis of nPHB in vitro: thermostable „activator“ and heat sensitive depolymerase (MERRICK & DOUDOROFF, 1964). Hydrolytic activity increased (> 6x) after preincubation of nPHB with activator or proteases. The activator was protease sensitive stable in alkanes, ketones, alcohols, slightly polar solvent. PhaZ1Rr (35 kDa, Topt. 50°C, pHopt. 9) hydrolyzed nPHB (3.1 mmol cuts min-1 mg-1) to 3-HA-mono-, di- and trimer independent of PHB-source and activation type. Neither lipase protease nor significant esterase activity (soluble esters) was found. Structural gene was expressed in E. coli. Amino acid sequence revealed homologies to dPHASCL depolymerases (type 2) but linker or substrate binding domains were absent. Lipase box (GIS19SG) consists with PHAMCL depolymerase. Sequence homologies and secondary structure analysis suggest a catalytical triad: S19, D115, H155 (H250G251 oxyanion)

The “Intracellular” Poly(3-Hydroxybutyrate) (PHB) Depolymerase of Rhodospirillum rubrum Is a Periplasm-Located Protein with Specificity for Native PHB and with Structural Similarity to Extracellular PHB Depolymerases

Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser(42), Asp(138), and His(178) were identified as catalytic-triad amino acids, with Ser(42) as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum

Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies.

Aiming at streamlining GPCR production from E. coli inclusion bodies for structural analysis, we present a generic approach to assess and optimize refolding yield through thermostability analysis. Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding, we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). Successful expression, purification and refolding is shown for two G protein-coupled receptors (GPCR), the sphingosine-1-phosphate receptor S1P1, and the orphan receptor GPR3. Refolded receptors were subjected to lipidic cubic phase crystallization screening