Prevalence and prognostic value of electrocardiographic abnormalities in hypokalemia: A multicenter cohort study.

BACKGROUND: Hypokalemia is common in hospitalized patients and associated with ECG abnormalities. The prevalence and prognostic value of ECG abnormalities in hypokalemic patients are, however, not well established. METHODS: The study was a multicentered cohort study, including all ault patients with an ECG and potassium level <4.4 mmol/L recorded at arrival to four emergency departments in Denmark and Sweden. Using computerized measurements from ECGs, we investigated the relationship between potassium levels and heart rate, QRS duration, corrected QT (QTc) interval, ST-segment depressions, T-wave flattening, and T-wave inversion using cubic splines. Within strata of potassium levels, we further estimated the hazard ratio (HR) for 7-day mortality, admission to the intensive care unit (ICU), and diagnosis of ventricular arrhythmia or cardiac arrest, comparing patients with and without specific ECG abnormalities matched 1:2 on propensity scores. RESULTS: Among 79,599 included patients, decreasing potassium levels were associated with a concentration-dependent increase in all investigated ECG variables. ECG abnormalities were present in 40% of hypokalemic patients ([K+ ] <3.5 mmol/L), with T-wave flattening, ST-segment depression, and QTc prolongation occurring in 27%, 16%, and 14%. In patients with mild hypokalemia ([K+ ] 3.0-3.4 mmol/L), a heart rate >100 bpm, ST-depressions, and T-wave inversion were associated with increased HRs for 7-day mortality and ICU admission, whereas only a heart rate >100 bpm predicted both mortality and ICU admission among patients with [K+ ] <3.0 mmol/L. HR estimates were, however, similar to those in eukalemic patients. The low number of events with ventricular arrhythmia limited evaluation for this outcome. CONCLUSIONS: ECG abnormalities were common in hypokalemic patients, but they are poor prognostic markers for short-term adverse events under the current standard of care.

In utero exposure to ADHD medication and long-term offspring outcomes.

Attention Deficit Hyperactivity Disorder (ADHD) medication is increasingly being used during pregnancy. Concerns have been raised as to whether ADHD medication has long-term adverse effects on the offspring. The authors investigated whether in utero exposure to ADHD medication was associated with adverse long-term neurodevelopmental and growth outcomes in offspring. The population-based cohort study in the Danish national registers included 1,068,073 liveborn singletons from 1998 to 2015 followed until any developmental diagnosis, death, emigration, or December 31, 2018. Children of mothers who continued ADHD medication (methylphenidate, amphetamine, dexamphetamine, lisdexamphetamine, modafinil, atomoxetine, clonidine) during pregnancy and children of mothers who discontinued ADHD medication before pregnancy were compared using Cox regression. Main outcomes were neurodevelopmental psychiatric disorders, impairments in vision or hearing, epilepsy, seizures, or growth impairment during childhood or adolescence. In total, 898 children were exposed to ADHD medication during pregnancy compared to 1270 children whose mothers discontinued ADHD medication before pregnancy. After adjustment for demographic and psychiatric characteristics of the mother, no increased risk of any offspring developmental disorders was found combined (aHR 0.97, 95% CI 0.81 to 1.17) or for separate subcategories. Similarly, no increased risk was found for any sub-categories of outcomes in the negative control or sibling controlled analyses. Neurodevelopment and growth in offspring do not differ based on antenatal exposure to ADHD medication. These findings provide reassurance for women with ADHD who depend on ADHD medication for daily functioning and who consider continuing medication in pregnancy.

Is the sequence ratio an unbiased estimate of the incidence rate ratio? A simulation study.

PURPOSE: We aimed to evaluate the conditions under which the sequence ratio (SR) obtained from a sequence symmetry analysis is an unbiased estimate of the true incidence rate ratio (IRR). METHODS: We simulated cohorts of 1 million individuals who could initiate an exposure drug and experience a very rare, rare, common, or frequent outcome of interest. The outcome rate among exposed individuals was modified by a true incidence rate ratio of 0.2, 0.5, 1.0, 2.0, and 5.0. We further evaluated scenarios where the outcome was fatal and led to immediate censoring or the outcome reduced the rate of initiation of the exposure drug. RESULTS: We found the SR to be close to unbiased for rare, common, and frequent events, except when the true IRR was 5.0 (mean SR 4.94 and 3.74 for common and frequent events). The SR was slightly biased when the outcome was very rare. When the outcome was potentially fatal, the SR was increasingly biased with an increasing probability of death. Likewise, when the outcome reduced the probability of future exposure, the SR was upwards biased. CONCLUSION: The SR is a biased estimate of the incidence rate ratio, when the true IRR is high, the outcome has a high mortality, or when the outcome reduces the probability of future exposure.

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening.

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium.

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine-the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfection of Gs-Enbrel and Aspg-Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5-fold higher specific productivity and ~3-fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.

Melatonin use among children, adolescents, and young adults: a Danish nationwide drug utilization study.

We aimed to provide a detailed description of the use of melatonin in Danish children, adolescents, and young adults during 2012-2019. We identified melatonin users 0-24 years of age (n = 43,652; median age 16 years) via the Danish nationwide health registers. Melatonin is a prescription drug in Denmark. The incidence of melatonin use increased from 2.4 to 3.9/1000 person-years during 2012 to 2019. Among 6,557 incident users in 2019, 53% filled only a single prescription within the first 6 months. Long-term use was most common among the younger age groups, with 17% of 5-9-year-olds and 14% of 10-13-year-olds being in continued treatment (no treatment breaks) 12 months after their first melatonin prescription. Disregarding treatment breaks, 3 in 10 were using melatonin 12 months after their first melatonin prescription and this proportion was also highest among 5-9-year-olds (63%) and 10-13-year-olds (51%). Psychopathology was common among melatonin users with 75% registered with either a psychiatric disorder diagnosis (54%), a filled prescription for another psychotropic (58%), or a contact to a private practice psychiatrist (15%) within ± 12 months of treatment initiation. General practitioners authorized melatonin prescriptions to almost half of all new users (48%), while psychiatric specialists authorized 37% of first prescriptions. In conclusion, the incidence of melatonin use increased in Denmark from 2012 to 2019. A substantial proportion of users had concurrent psychopathology most likely explaining their use of melatonin. Long-term melatonin use was more common among the youngest age groups, which should be a focus of interest due to limited safety data.

Changing characteristics over time of individuals receiving COVID-19 vaccines in Denmark: A population-based descriptive study of vaccine uptake.

AIMS: The Danish authorities implemented a differential rollout of the COVID-19 vaccines where individuals at high risk of COVID-19 were prioritized. We describe the temporal uptake and characteristics of COVID-19 vaccine recipients in Denmark. METHODS: Using nationwide healthcare registries, we identified all Danish residents ⩾5 years of age who received at least one dose of a COVID-19 vaccine from 27 December 2020-29 January 2022. We charted the daily number of newly vaccinated individuals and the cumulative vaccine coverage over time, stratified by vaccine type, age groups and vaccination priority groups, and described characteristics of vaccine recipients during two-month-intervals and in vaccination priority groups. RESULTS: By 29 January 2022, 88%, 86% and 64% of Danish residents ⩾5 years (n=5,562,008) had received a first, second and third dose, respectively, of a COVID-19 vaccine, most commonly the BNT162b2 vaccine (84%). Uptake ranged from 48% in 5-11-year-olds to 98% in 65-74-year-olds. Individuals vaccinated before June 2021 were older (median age 61-70 years vs 10-35 years in later periods) and had more comorbidities such as hypertension (22-28% vs 0.77-2.8% in later periods), chronic lung disease (9.4-15% vs 3.7-4.6% in later periods) and diabetes (9.3-12% vs 0.91-2.4% in later periods). CONCLUSIONS: We document substantial changes over time in, for example, age, sex and medical history of COVID-19 vaccine recipients. Though these results are related to the differential vaccine rollout in Denmark, similar findings are probable in other countries and should be considered when designing and interpreting studies on the effectiveness and safety of COVID-19 vaccines.

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism.

Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures.

Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production. Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells. Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2-44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode.

Awakening dormant glycosyltransferases in CHO cells with CRISPRa.

Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced. This study demonstrates that CRISPRa can make targeted alterations of CHO cells for desired phenotypes.

Modular 5'-UTR hexamers for context-independent tuning of protein expression in eukaryotes.

Functional characterization of regulatory DNA elements in broad genetic contexts is a prerequisite for forward engineering of biological systems. Translation initiation site (TIS) sequences are attractive to use for regulating gene activity and metabolic pathway fluxes because the genetic changes are minimal. However, limited knowledge is available on tuning gene outputs by varying TISs in different genetic and environmental contexts. Here, we created TIS hexamer libraries in baker's yeast Saccharomyces cerevisiae directly 5' end of a reporter gene in various promoter contexts and measured gene activity distributions for each library. Next, selected TIS sequences, resulted in almost 10-fold changes in reporter outputs, were experimentally characterized in various environmental and genetic contexts in both yeast and mammalian cells. From our analyses, we observed strong linear correlations (R2 = 0.75-0.98) between all pairwise combinations of TIS order and gene activity. Finally, our analysis enabled the identification of a TIS with almost 50% stronger output than a commonly used TIS for protein expression in mammalian cells, and selected TISs were also used to tune gene activities in yeast at a metabolic branch point in order to prototype fitness and carotenoid production landscapes. Taken together, the characterized TISs support reliable context-independent forward engineering of translation initiation in eukaryotes.

Reprogramming AA catabolism in CHO cells with CRISPR/Cas9 genome editing improves cell growth and reduces byproduct secretion.

Chinese hamster ovary (CHO) cells are the preferred host for producing biopharmaceuticals. Amino acids are biologically important precursors for CHO metabolism; they serve as building blocks for proteogenesis, including synthesis of biomass and recombinant proteins, and are utilized for growth and cellular maintenance. In this work, we studied the physiological impact of disrupting a range of amino acid catabolic pathways in CHO cells. We aimed to reduce secretion of growth inhibiting metabolic by-products derived from amino acid catabolism including lactate and ammonium. To achieve this, we engineered nine genes in seven different amino acid catabolic pathways using the CRISPR-Cas9 genome editing system. For identification of target genes, we used a metabolic network reconstruction of amino acid catabolism to follow transcriptional changes in response to antibody production, which revealed candidate genes for disruption. We found that disruption of single amino acid catabolic genes reduced specific lactate and ammonium secretion while specific growth rate and integral of viable cell density were increased in many cases. Of particular interest were Hpd and Gad2 disruptions, which show unchanged AA uptake rates, while having growth rates increased up to 19%, and integral of viable cell density as much as 50% higher, and up to 26% decrease in specific ammonium production and to a lesser extent (up to 22%) decrease in lactate production. This study demonstrates the broad potential of engineering amino acid catabolism in CHO cells to achieve improved phenotypes for bioprocessing.

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles.

Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest. Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms.

The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality. With the growing toolbox of genetic engineering technologies, it is now possible to address general as well as host- or biopharmaceutical-specific product quality obstacles. In this review, we present diverse expression systems derived from mammalians, bacteria, yeast, plants, and insects as well as available genetic engineering tools. We focus on genes for knockout/knockdown and overexpression for meaningful approaches to improve biopharmaceutical PTMs and discuss their applicability as well as future trends in the field.

Reduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interference.

Chinese hamster ovary (CHO) cells are widely used for biopharmaceutical protein production. One challenge limiting CHO cell productivity is apoptosis stemming from cellular stress during protein production. Here we applied CRISPR interference (CRISPRi) to downregulate the endogenous expression of apoptotic genes Bak, Bax, and Casp3 in CHO cells. In addition to reduced apoptosis, mitochondrial membrane integrity was improved and the caspase activity was reduced. Moreover, we optimized the CRISPRi system to enhance the gene repression efficiency in CHO cells by testing different repressor fusion types. An improved Cas9 repressor has been identified by applying C-terminal fusion of a bipartite repressor domain, KRAB-MeCP2, to nuclease-deficient Cas9. These results collectively demonstrate that CHO cells can be rescued from cell apoptosis by targeted gene repression using the CRISPRi system.

A Markov chain model for N-linked protein glycosylation--towards a low-parameter tool for model-driven glycoengineering.

Glycosylation is a critical quality attribute of most recombinant biotherapeutics. Consequently, drug development requires careful control of glycoforms to meet bioactivity and biosafety requirements. However, glycoengineering can be extraordinarily difficult given the complex reaction networks underlying glycosylation and the vast number of different glycans that can be synthesized in a host cell. Computational modeling offers an intriguing option to rationally guide glycoengineering, but the high parametric demands of current modeling approaches pose challenges to their application. Here we present a novel low-parameter approach to describe glycosylation using flux-balance and Markov chain modeling. The model recapitulates the biological complexity of glycosylation, but does not require user-provided kinetic information. We use this method to predict and experimentally validate glycoprofiles on EPO, IgG as well as the endogenous secretome following glycosyltransferase knock-out in different Chinese hamster ovary (CHO) cell lines. Our approach offers a flexible and user-friendly platform that can serve as a basis for powerful computational engineering efforts in mammalian cell factories for biopharmaceutical production.

Glycoprofiling effects of media additives on IgG produced by CHO cells in fed-batch bioreactors.

Therapeutic monoclonal antibodies (mAbs) are mainly produced by heterologous expression in Chinese hamster ovary (CHO) cells. The glycosylation profile of the mAbs has major impact on the efficacy and safety of the drug and is therefore an important parameter to control during production. In this study, the effect on IgG N-glycosylation from feeding CHO cells with eight glycosylation precursors during cultivation was investigated. The study was conducted in fed-batch mode in bioreactors with biological replicates to obtain highly controlled and comparable conditions. We assessed charge heterogeneity and glycosylation patterns of IgG. None of the eight feed additives caused statistically significant changes to cell growth or IgG productivity, compared to controls. However, the addition of 20 mM galactose did result in a reproducible increase of galactosylated IgG from 14% to 25%. On the other hand, addition of 20 mM N-acetyl-D-glucosamine (GlcNAc) reduced relative abundance of galactosylated IgG by 4%. Additionally, supplementation with 10 mM mannose slightly reduced GlcNAc occupancy of IgG. Overall, comparing the effects of IgG glycosylation, by supplementing the cell culture medium with glycosylation precursors during cultivation, revealed an application of these glycosylation precursors for modulating N-glycosylation of IgG.

Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment.

Targeted gene integration into site-specific loci can be achieved in Chinese hamster ovary (CHO) cells via CRISPR/Cas9 genome editing technology and the homology-directed repair (HDR) pathway. The low efficiency of HDR often requires antibiotic selection, which limits targeted integration of multiple genes at multiple sites. To improve HDR-mediated targeted integration, while avoiding the use of selection markers, chemical treatment for increased HDR, and fluorescent enrichment of genome-edited cells was assessed in CHO cells. Chemical treatment did not improve HDR-mediated targeted integration. In contrast, fluorescent markers in Cas9 and donor constructs enable FACS enrichment, resulting in a threefold increase in the number of cells with HDR-mediated genome editing. Combined with this enrichment method, large transgenes encoding model proteins (including an antibody) were successfully targeted integrated. This approach provides a simple and fast strategy for targeted generation of stable CHO production cell lines in a rational way. Biotechnol. Bioeng. 2016;113: 2518-2523. © 2016 Wiley Periodicals, Inc.