Stable HEK293 Cell Research Articles

Rapid and Scalable Production of Functional SARS-CoV-2 Virus-like Particles (VLPs) by a Stable HEK293 Cell Pool.

At times of pandemics, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the situation demands rapid development and production timelines of safe and effective vaccines for delivering life-saving medications quickly to patients. Typical biologics production relies on using the lengthy and arduous approach of stable single-cell clones. Here, we used an alternative approach, a stable cell pool that takes only weeks to generate compared to a stable single-cell clone that needs several months to complete. We employed the membrane, envelope, and highly immunogenic spike proteins of SARS-CoV-2 to produce virus-like particles (VLPs) using the HEK293-F cell line as a host system with an economical transfection reagent. The cell pool showed the stability of protein expression for more than one month. We demonstrated that the production of SARS-CoV-2 VLPs using this cell pool was scalable up to a stirred-tank 2 L bioreactor in fed-batch mode. The purified VLPs were properly assembled, and their size was consistent with the authentic virus. Our particles were functional as they specifically entered the cell that naturally expresses ACE-2. Notably, this work reports a practical and cost-effective manufacturing platform for scalable SARS-CoV-2 VLPs production and chromatographic purification.

Abstract 2065: Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity

Abstract Adenosine Deaminase Acting on RNA (ADAR) enzymes perform adenosine to inosine base editing in RNA, particularly targeting adenosines located within a specific double-stranded stem-loop motif. The ADAR1 gene encodes both a universally expressed isoform (p110) and an interferon-inducible isoform (p150), which plays a role in innate immunity by mediating interferon signaling. In the context of healthy, uninfected cells, ADAR1 performs A-to-I editing on endogenous double-stranded RNA to prevent it from activating downstream dsRNA sensors RIG-I and MDA5, which in-turn activate a pro-inflammatory response. Loss of function mutations in ADAR1 result in aberrant activation of the dsRNA sensors and are involved in autoimmune disorders. ADAR1 dysfunction also impacts cancer cell growth, proliferation, and response to immunotherapy. ADAR1 expression is increased in several types of cancer and ADAR1 knock-out has been demonstrated to improve the response to certain immunotherapies such PD-1/PD-L1 blockade and to circumvent tumor immunotherapy resistance mechanisms, making ADAR1 an attractive target for therapeutic development. We have developed and validated a high-throughput, cell-based assay for measuring ADAR1 editing activity. Several ADAR editing reporter constructs were designed and evaluated for their relative response to ADAR1 over-expression. They all feature an ADAR1 hairpin target with a stop codon (UAG) which is susceptible to ADAR1-mediated editing to a tryptophan (UUG), located upstream of a firefly luciferase gene. We show that increasing transient expression of ADAR1 led to proportional increased activity of the downstream firefly luciferase, indicating dose-dependent transcript editing by ADAR1. Selected reporters were used in conjunction with ADAR2 overexpression to identify an ADAR1-biased reporter. Thus, a reporter with low luciferase activity in response to ADAR2 expression was chosen to establish stable HEK293 cell lines (which express low levels of ADAR1) with and without constitutively expressed ADAR1 protein. ADAR1-overexpressing HEK293 cells displayed high luciferase activity, in accordance with high ADAR1-mediated editing of the reporter transcript, which was decreased in response to siRNA-mediated knockdown of ADAR1 expression or pharmacological inhibition of ADAR1 activity. Sequencing of the reporter RNA confirmed that changes in reporter activity were correlated with editing of the RNA transcript. Finally, these validated ADAR1 editing reporter cell lines were further engineered to constitutively express Renilla luciferase under the control of a CMV promoter, serving as an internal control for the toxicity of ADAR1 inhibitors. In conclusion, our robust cell-based assay provides a well-validated resource to identify modulators of ADAR1 editing activity in a cellular context. Citation Format: Valerie Sapp, Jim Bilakovics, Ava Burr, Fernando Martins, Pavel Shashkin, Veronique Baron, Henry Zhu, Junguk Park. Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2065.

Development and application of a multiple reaction monitoring method for the simultaneous quantification of sodium channels Nav1.1, Nav1.2, and Nav1.6 in solubilized membrane proteins from stable HEK293 cell lines, rodents, and human brain tissues

RationaleNav1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage‐gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development.MethodsUnique peptides for Nav1.1, Nav1.2, and Nav1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography–electrospray ionization tandem mass spectrometry.ResultsNav 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/μg membrane protein, respectively. In brains, Nav1.2 showed the highest expression, with approximately three times higher (P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/μg in human brain. Both Nav1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Nav1.1 than rodent Nav1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Nav1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/μg membrane proteins.ConclusionsMultiple reaction monitoring was used to quantify sodium channels Nav1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Nav expression ranking among the three species was Nav1.2 ≫ Nav1.1 > Nav1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.

High-throughput ligand profile characterization in novel cell lines expressing seven heterologous insect olfactory receptors for the detection of volatile plant biomarkers

Agriculturally important crop plants emit a multitude of volatile organic compounds (VOCs), which are excellent indicators of their health status and their interactions with pathogens and pests. In this study, we have developed a novel cellular olfactory panel for detecting fungal pathogen-related VOCs we had identified in the field, as well as during controlled inoculations of several crop plants. The olfactory panel consists of seven stable HEK293 cell lines each expressing a functional Drosophila olfactory receptor as a biosensing element along with GCaMP6, a fluorescent calcium indicator protein. An automated 384-well microplate reader was used to characterize the olfactory receptor cell lines for their sensitivity to reference VOCs. Subsequently, we profiled a set of 66 VOCs on all cell lines, covering a concentration range from 1 to 100 μM. Results showed that 49 VOCs (74.2%) elicited a response in at least one olfactory receptor cell line. Some VOCs activated the cell lines even at nanomolar (ppb) concentrations. The interaction profiles obtained here will support the development of biosensors for agricultural applications. Additionally, the olfactory receptor proteins can be purified from these cell lines with sufficient yields for further processing, such as structure determination or integration with sensor devices.

The generation of detergent-insoluble clipped fragments from an ERAD substrate in mammalian cells

Proteostasis ensures the proper synthesis, folding, and trafficking of proteins and is required for cellular and organellar homeostasis. This network also oversees protein quality control within the cell and prevents accumulation of aberrant proteins, which can lead to cellular dysfunction and disease. For example, protein aggregates irreversibly disrupt proteostasis and can exert gain-of-function toxic effects. Although this process has been examined in detail for cytosolic proteins, how endoplasmic reticulum (ER)-tethered, aggregation-prone proteins are handled is ill-defined. To determine how a membrane protein with a cytoplasmic aggregation-prone domain is routed for ER-associated degradation (ERAD), we analyzed a new model substrate, TM-Ubc9ts. In yeast, we previously showed that TM-Ubc9ts ERAD requires Hsp104, which is absent in higher cells. In transient and stable HEK293 cells, we now report that TM-Ubc9ts degradation is largely proteasome-dependent, especially at elevated temperatures. In contrast to yeast, clipped TM-Ubc9ts polypeptides, which are stabilized upon proteasome inhibition, accumulate and are insoluble at elevated temperatures. TM-Ubc9ts cleavage is independent of the intramembrane protease RHBDL4, which clips other classes of ERAD substrates. These studies highlight an unappreciated mechanism underlying the degradation of aggregation-prone substrates in the ER and invite further work on other proteases that contribute to ERAD.

SAT025 Characterization Of The Melatonin Type 1 Receptor / Cannabinoid Type 1 Receptor (MT1/CB1) Heteromers

Abstract Disclosure: Y. Cui: None. H. Cherdoud: None. G. Marsicano: None. L. Bellocchio: None. R. Jockers: None. O. Lahuna: None. Membrane receptors belonging to the G proteins coupled receptors (GPCRs), the largest family of proteins in the human genome with more than 800 members. GPCR functions were initially thought to take place at the plasma membrane upon binding of single monomeric receptor unit by their cognate ligand. This scheme was complexified after identification of cross-talks between GPCRs upon heteromerization and identification of functional GPCRs within intracellular organelles as nucleus or endosomes. Among a few others receptors, functional melatonin receptor type 1 (MT1) and cannabinoid receptor type 1 (CB1) were found in neuronal mitochondria, suggesting a cross-talk between them. Here, we are identifying and characterizing MT1/CB1 heteromers as a new GPCR heteromer. Previously we showed that coexpression of the MT1 and CB1 receptors induced the constitutive formation of MT1/CB1 heteromers using coimmunoprecipitation experiments on transiently transfected HEK293 cells. Using simultaneous PLA (Proximity Ligation Assay) and immunofluorescence experiments, we described the existence of intracellular MT1/CB1 heteromers associated with the mitochondrial network. Since then, we have further characterized the properties of MT1/CB1 heteromers. Coimmunoprecipitation experiments performed on cerebellum from wild-type, MT1 or CB1 knock-out mice enabled us to identify MT1/CB1 heteromers in vivo. We established stable monoclonal HEK293 cell lines expressing both receptors. Transmission Electronic Microscopy (TEM) and PLA experiments showed the presence of MT1/CB1 complexes not only intracellularly but also at the cell surface. Using PLA experiments we identified a penetrating interfering peptide that specifically dissociate the MT1/CB1 complex and thus open the door to experiments targeting specifically the complex. We were able to follow the melatonin-dependent internalization and trafficking of the MT1/CB1 complex from the plasma membrane to the late endosomes using a combination of Nanoluciferase complementation experiments and bystander BRET (Bioluminescence Resonance Energy Transfer). Costimulation of HE293 cells expressing both receptors with melatonin and the selective CB1 agonist ACEA (Arachidonyl-2'-chloroethylamide), a derivative of the endocannabinoid AEA (Arachidonylethanolamide or anandamide) resulted in a potentiation of the melatonin-dependent cAMP inhibition. Use of the identified interfering peptide will enable us to identify actions on signaling pathways resulting from a direct cross-talk between MT1 or CB1 subunits. Presentation: Saturday, June 17, 2023

Characterization of a monoclonal antibody specific to California serogroup orthobunyaviruses and development as a chimeric immunoglobulin M-positive control in human diagnostics.

Orthobunyaviruses in the California serogroup cause severe neurological disease in children and adults. While these viruses are known to circulate widely in North America, their occurrence is rare. Serological testing for CSGVs is hindered by the limited availability and volumes of human-positive specimens needed as controls in serologic assays. Here, we described the development of a murine monoclonal antibody cross-reactive to CSGVs engineered to contain the variable regions of the murine antibody on the backbone of human IgM. The chimeric IgM produced from the stably expressing HEK293 cell line was evaluated for use as a surrogate human-positive control in a serologic diagnostic test.

CL-705G: a novel chemical Kir6.2-specific KATP channel opener.

Background: KATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various KATP channel subclasses. Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked "hits", which were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 of 9µM and 11μM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na+ currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism. Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.

Characterization of expanded γδ T cells from patient with atypical X-linked severe combined immunodeficiency reveals preserved function and IL2RG-mediated signaling

Abnormally high γδ T cell numbers are common among atypical severe combined immunodeficiency (SCID) patients but detailed immunophenotyping and functional characterization of these expanded γδ T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG c.172C > T;p.(Pro58Ser) variant causing failure in IL2RG (CD132) plasma membrane targeting leading to impaired IL-2 responses in CD4 and CD8 T cells. Our aim was to investigate the phenotype and function of the index patient's abnormally large peripheral γδ T cell population.We found that in contrast to his αβ T cells, the patient's γδ T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. Vδ2+ population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's γδ T cells showed enhanced cytotoxicity towards A549 cancer cells. TCRvγ repertoire was versatile but sequencing of IL2RG revealed a novel c.534C>A; p.(Phe178Leu) somatic missense variant restricted to γδ T cells. Over time this variant became predominant in patient's γδ T cells and was enriched during in vitro expansion of patient's γδ T cells, indicating that the clones with this somatic variant may have growth or survival advantage. In silico analysis with DynaMut2 tool indicated that the overall effect of Phe178Leu substitution for IL2RG was predicted to be stabilizing and IL2RG-Pro58Ser/Phe178Leu to be more stable than IL2RG-Pro58Ser variant alone. Furthermore, IL2RG-Pro58Ser/Phe178Leu showed significantly increased cell surface expression when compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic IL2RG p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline IL2RG p.(Pro58Ser) variant.In conclusion, our results indicate that expansion of γδ T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells. (Tuovinen et al., Journal of Clinical Immunology 2022)

PO-04-166 DISTURBED PH CONTROL BY A NOVEL SLC4A3 VARIANT IDENTIFIED IN A SHORT QT SYNDROME FAMILY WITH A KCNH2 VARIANT

Short QT syndrome (SQTS) is one of the inherited primary arrhythmia syndromes and characterized by shortened QT intervals leading to ventricular fibrillation (VF) and sudden cardiac death, especially in young people. The major cause of SQTS is pathogenic variants in KCNH2 encoding Kv11.1 which produce IKr. Recently, a novel variant in SLC4A3 encoding the anion exchanger (AE3) has been identified in two SQTS families. However, the pathogenesis of SQTS caused by SLC4A3 variants in the presence of a KCNH2 variant have not been elucidated yet. We aimed to elucidate the functional change of a novel SLC4A3 variant in the interaction with a KCNH2 variant. We performed targeted gene sequencing (TGS) and whole exome sequencing (WES) in SQTS patients. Electrophysiological characteristics of the KCNH2 variant were analyzed using HEK293 cells by patch-clamp methods. We constructed stable HEK293 cell lines expressing SLC4A3 with or without the variant and measured intracellular pH (pHi) by ratiometric pH indicator, BCECF-AM. Using the stable cell lines, we transfected KCNH2 (WT or the variant), KCNQ1 and KCNJ2 and measured IKr, IKs and IK1. We identified a KCNH2 variant, c.280 c>t, p.H70Y by TGS in a 25-year-old man who suffered VF at night and was diagnosed with SQTS after resuscitation. We first performed electrophysiological analysis of KCNH2-H70Y alone, however, the variant affected a little of Ikr properties. Therefore, we performed WES in the proband and his family, and a novel SLC4A3 variant, c.1059 c>a, p.N353K was identified. Both variants were co-segregated in his family with short QT intervals. In the live cell imaging analysis, cells expressing SLC4A3-N353K showed significantly slower pHi alternations than those with WT (figure A), reflecting the loss-of-function effect of exchange kinetics in mutant AE3. In the electrophysiological analysis using stable cell lines expressing SLC4A3, IKs and IK1 did not show any changes in channel properties regardless the presence of SLC4A3-N353K. However, IKr with H70Y showed significantly slow deactivation time constant in the cells expressing SLC4A3-N353K (Figure B and C), which means the gain-of-function effect on Kv11.1. SLC4A3-N353K showed slower alteration of pHi, a loss-of-function effect on AE3. With the expression of SLC4A3-N353K,KCNH2-H70Y decelerated the deactivation kinetics of Ikr, which may lead to shortening of QT intervals in our patient.

A novel KIR-specific KATP channel opener.

KATP channels have diverse roles, including regulation of insulin secretion, protection against cardiac stress responses, and blood flow regulation, and are therefore excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the specific assemblies of pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against various KATP channel subclasses. We generated 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures in both the open and closed states and identified a potential agonist binding pocket in a functionally critical area of the channel in the vicinity of a palmitoylated Kir6.2-C166 residue and the PIP2 binding region. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds were performed. The top 15 ‘hits’ were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes and of them (70862985) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 respectively 5 µM and 1 µM). Remarkably, at 15-100 µM, compound 70862985 had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1 or Kir3.1/Kir3.4 channels and at 100 µM minimally affected Na+ currents in TE671 medulloblastoma cells. We examined whether 70862985 can correct Kir6.2 mutants with a decreased open probability, which is typically associated with congenital hyperinsulinism. Each of the Kir6.2-F55L, Kir6.2-T62M and Kir6.2-R301C mutants were associated with reduced Tl+ fluxes from Kir6.2/SUR1 transfected HEK-293 cells and were relatively insensitive to diazoxide. Kir6.2-R301C, by contrast, was strongly activated by 70862985. 70862985 is a Kir6.2 opener that does not affect the structurally similar Kir6.1 channel. This, to our knowledge, is the first Kir-specific channel opener.

PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells

Although mounting evidence has demonstrated that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) can promote tumorigenesis, its role in cancer remains controversial. To find potential target molecules of PGC-1α, GeneFishingTM DEG (differentially expressed genes) screening was performed using stable HEK293 cell lines expressing PGC-1α (PGC-1α-HEK293). As results, leucyl-tRNA synthetase 1 (LARS1) was upregulated. Western blot analysis showed that LARS1 was increased in PGC-1α overexpressed SW480 cells but decreased in PGC-1α shRNA knockdown SW620 cells. Several studies have suggested that LARS1 can be a potential target of anticancer agents. However, the molecular network of PGC-1α and LARS1 in human colorectal cancer cells remains unclear. LARS1 overexpression enhanced cell proliferation, migration, and invasion, whereas LARS1 knockdown reduced them. We also observed that expression levels of cyclin D1, c-Myc, and vimentin were regulated by LARS1 expression. We aimed to investigate whether effects of PGC-1α on cell proliferation and invasion were mediated by LARS1. Our results showed that PGC-1α might modulate cell proliferation and invasion by regulating LARS1 expression. These results suggest that LARS1 inhibitors might be used as anticancer agents in PGC-1α-overexpressing colorectal cancer. Further studies are needed in the future to clarify the detailed molecular mechanism by which PGC-1α regulates LARS1 expression.

Mutational Permutations of a Single Amino Acid Exert Divergent Phenotypic Effects in Human, Mice, and Cellular Models of SAMD9L Bone Marrow Failure Disorder

While characterizing the new SAMD9 and SAMD9L - related bone marrow failure syndromes, we found that different germline pathogenic mutations can occur at the same amino acid position (Sahoo, Nat, Medicine 2021). Here, we explore this phenomenon by studying a family with triple allelic mosaicism in SAMD9L and two mutational permutations of the same amino acid in different children. We applied genomic methods (exome sequencing, Sanger, error-corrected sequencing, FISH, single-cell DNA sequencing (scDNAseq), ddPCR, and PacBio haplotype sequencing), developed stable mutant HEK293 cell lines, created V1512M and V1512Lmutant inducible pluripotent stem cells (iPSC) and ortholog mutant mouse models with Samd9l heterozygous (V1507Mhet, V1507Lhet) and homozygous (V1507Lhom) mutations using CRISPR/Cas9 engineering. In a family with SAMD9L-related bone marrow failure, the affected girl carried heterozygous c.4534G>A (V1512M) mutation, while her brother was heterozygous for c.4534G>T (V1512L) mutation. Both mutations were germline and confirmed to arise from the same haplotype. The child with V1512M also carried somatic rescue events (del7q, acquired SAMD9L mutation) and had a more severe clinical phenotype. The asymptomatic mother who had normal diploid karyotype was a triple-allelic mosaic. The SAMD9L wildtype (WT) allele was present at ~50% frequency in all tissues, while both mutant alleles present in the remaining 50% of reads "competed" in their allelic distribution depending on the tissue origin (hematopoietic cells: V1512M ~9% and V1512L ~41%, hair follicles: both at ~25%). ScDNAseq revealed 3 separate clonal populations in blood, all with biallelic configuration: SAMD9L V1512M in 14%, V1512L in 68%, and WT (with acquired uniparental isodisomy 7q) in 18% of cells. Because her parents were SAMD9L WT, one of the mutations likely arose de novo and underwent failed rescue attempt during embryogenesis leading to a second mutation. Overexpression studies in HEK293 cells demonstrated significant growth inhibition of mutants (V1512M more severe than V1512L) compared to WT. Hematopoietic progenitor cells (HPC) derived from mutated iPSC had severely compromised proliferative capacity, and differentiation of V1512M and V1512L HPC yielded significantly fewer CD235+CD71+ erythroid cells and severely decreased CD45+CD33+ myeloid cells; in both lineages, V1512M mutation had the most severe phenotype. We also observed divergent mutational phenotypes in mice: two-thirds (13/19) of the Samd9l V1507Mhet pups died before weaning and the vast majority died within the first 2 weeks of life. On the contrary, V1507Lhet mice had overall survival equal to WT mice, while V1507Lhom mice had a trend of decreased survival with 40% of mice dying by 19 weeks. At baseline, Samd9l V1507Mhet mice showed severe growth retardation with multisystem involvement (kidney, ovary, and alimentary tract), while V1507Lhet and V1507Lhom mice had no such abnormalities. The histology of BM and spleen in >7 weeks old V1507Mhet mice showed decreased erythroid and increased myeloid precursors in BM with extramedullary hematopoiesis. Significant multi-lineage peripheral blood (PB) cytopenia were observed in V1507Mhet mice compared to WT littermates. V1507Lhet mice had normal PB counts and BM phenotype comparable to WT, while V1507Lhom mice had isolated cytopenia and reduced numbers of BM erythroid and B-cells, and an increase in myeloid cells. Next, to induce replicative stress in V1507Lhom model, we performed non-competitive BM transplantation (BMT) into lethally irradiated B6.SJL/J WT recipients. At 12 weeks post BMT, there was a significant reduction of total V1507Lhom cell numbers with a decrease in B-cells, increase in myeloid cells, and no difference in T-cells. For V1507Mhet mice, we performed competitive BMT (because of the severe baseline phenotype), revealing significant changes post BMT, like V1507Lhom transplants, yet more severe and arising earlier. Altogether, our data suggest that Samd9l V1507Mhet and V1507Lhom cells have lower erythroid and B-cell output and a shift towards myelopoiesis. To our knowledge, this is the first report of germline triple-allelic mosaicism with bi-allelic segregation in humans. Our study establishes how different mutational permutations of the same amino acid exert divergent phenotypic effects in humans, mice, and cellular models of SAMD9L bone marrow failure disorder.

Characterization of Expanded Gamma Delta T Cells from Atypical X-SCID Patient Reveals Preserved Function and IL2RG-Mediated Signaling

Abnormally high γδ T cell numbers among individuals with atypical SCID have been reported but detailed immunophenotyping and functional characterization of these expanded γδ T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG (NM_000206.3) c.172C > T;p.(Pro58Ser) variant. Here, we have further investigated the index patient’s abnormally large γδ T cell population in terms of function and phenotype by studying IL2RG cell surface expression, STAT tyrosine phosphorylation and blast formation in response to interleukin stimulation, immunophenotyping, TCRvγ sequencing, and target cell killing. In contrast to his ⍺β T cells, the patient’s γδ T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. Vδ2 + population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient’s γδ T cells showed enhanced cytotoxicity towards A549 cancer cells. His TCRvγ repertoire was versatile but sequencing of IL2RG revealed a novel c.534C > A; p.(Phe178Leu) somatic missense variant restricted to γδ T cells. Over time this variant became predominant in γδ T cells, though initially present only in part of them. IL2RG-Pro58Ser/Phe178Leu variant showed higher cell surface expression compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline p.(Pro58Ser) variant. In conclusion, our report indicates that expansion of γδ T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells.

Probing the Central Role of Phosphatidylinositol Synthesis in Lipid Metabolism of Eukaryotic Cells

Phosphatidylinositol (PI) is the precursor of all phosphoinositides (PPIns), a class of regulatory phospholipids that play key roles in cellular physiology. PI is synthesized in the ER by a single enzyme, phosphatidylinositol synthase (PIS). PI may have important roles of its own in membranes where PPIns are not produced such as in the ER and mitochondria. Disruption of PIS in yeast and zebrafish is lethal and PIS has been postulated to be an essential gene in studies of haploid cells. Therefore, it is difficult to evaluate the processes where this enzyme, or its lipid product, PI plays a critical role. To overcome this problem, we generated stable HEK293 cell lines in which a doxycycline (Dox)‐inducible PIS‐GFP transgene was stably integrated in a safe‐harbor site of the genome and the endogenous PIS locus was inactivated via CRISPR/Cas9 genome editing. We verified the knock‐out of the endogenous PIS and the inducible expression of PIS‐GFP by western blot analysis and sequencing of genomic DNA. Using these cells, we found that PIS enzymatic activity assayed in ER membranes prepared from cells after Dox deprivation was indistinguishable from the assay blank. Dox treatment of cells elevated PIS activity in the isolated ER membrane although it was still lower than that found in the membranes prepared from parental HEK293. Surprisingly, in spite of the undetectable low PIS activity, Dox‐deprived cells still contained about 50% of PI as assessed by equilibrium labeling with 3H‐inositol and retained their PPIns distribution as shown by confocal microscopy using GFP‐tagged PPIns binding modules. This disparity between PIS activity and PI availability was observed even under culture conditions that deprived cells of any external PI source. Such cells were subjected to lipidomic analysis to assess the overall lipid changes associated with the reduction of PI synthesis and to identify processes that require PI. This analysis confirmed the ~50% PI loss and also revealed reprogramming of specific lipid metabolic pathways. Current work focuses on the possibility that alternate source(s) of PI exist and on the question of what lipid synthetic pathways show adaptation to the greatly reduced or missing PIS activity. These experiments have the promise of a better understanding of how PI lipids determine the overall lipid landscape and homeostasis of eukaryotic cells and may unravel new molecular targets that can be exploited for therapeutic interventions in human metabolic and lipid disorders.

Abstract 11857: Benchmarking a Functional Assay to Assist Clinical Classification of Variants of Uncertain Significance in KCNH2 (Long QT Syndrome Type 2)

Introduction: Our ability to determine the pathogenicity of variants is limited by the paucity of clinical data when variants are seen in very few individuals. In vitro functional assays could help overcome the deluge of variants of uncertain significance (VUS). To be clinically useful, such assays must satisfy stringent benchmarking criteria established by the ACMG/AMP. For variants in KCNH2 , the cause of Long QT Syndrome Type 2 (LQTS2), ~70% of missense variants are VUS. Here, we wish to formally evaluate the accuracy, replicability and robustness of a high-throughput automated patch-clamp (APC) assay for characterising KCNH2 variants. Aim: To evaluate an APC assay for KCNH2 variants against the criteria for the application of functional evidence within the ACMG/AMP framework. Methods: Twenty five variants classified as pathogenic/likely pathogenic and 18 variants classified as benign/likely benign from ClinVar were heterozygously expressed in stable Flp-In HEK293 cells and analysed for loss-of-function based on current density and changes in channel gating. Results: Twenty three of 25 pathogenic variants had <50% wild-type (WT) peak current density, indicating loss-of-function. The two remaining pathogenic variants, G584S and N629S, had <50% WT current levels (IKr) after incorporating additional gating measures. All 18 benign variants had >50% WT current levels both before and after including gating measures. For 17 of the benign variants, current levels were not statistically different to WT. The current level for R148W was significantly different to WT (p<0.005) but was still >50%. Based on a 50% cut-off, the assay yields a sensitivity of 100% and specificity of 100%. Conclusions: High-throughput electrophysiological phenotyping correctly predicted loss of function for 25/25 pathogenic variants, and 18/18 benign variants. The assay should be able to assist with the clinical classification of KCNH2 variants.

Employing the Operational Model to Measure System-Independent OTR Efficacy.

Ideally, pharmacological analysis is based on quantitative measurements applied toward unveiling biological mechanisms and guiding medicinal chemistry efforts in drug discovery and basic research. The most robust analysis frameworks generate findings which can be dissociated from the specific experimental setting to provide insights of a broader scope. With insufficient efficacy being the leading cause of drug failures in late-stage clinical trials, more rigorous preclinical definitions might assist in better translation. This chapter details a new method for accessing the Black and Leff operational model of agonism using a stable Flp-In™ T-REx™ HEK293 cell line under tetracycline-dependent control of OTR expression. We cover steps for performing the Gq-coupled HTRF® IP-One assay, curve-fitting data, calculating and statistically representing system-independent drug activity, predicting responses in different sensitivities, and plotting of multivariate analyses.

Fed-batch production assessment of a tetravalent bispecific antibody: A case study on piggyBac stably transfected HEK293 cells

The transition from preclinical biological drug development into clinical trials requires an efficient upscaling process. In this context, bispecific antibody drugs are particularly challenging due to their propensity to form aggregates and generally produce low titers. Here, the upscaling process for a tetravalent bispecific antibody expressed by a piggyBac transposon-mediated stable HEK293 cell pool has been evaluated. The project was performed as a case study at Testa Center, a non-GMP facility for scale-up testing of biologics in Sweden, and encompassed media adaptation strategies, fed-batch optimization and a novel antibody purification technology. The cell pool was adapted to different culture media for evaluation in terms of cell viability and titers compared to its original Expi293 Expression Medium. These parameters were assessed in both sequential stepwise adaption and direct media exchanges. By this, a more affordable medium was identified that did not require stepwise adaptation and with similar titers and viability as in the Expi293 Expression Medium. Fed-batch optimizations resulted in culture densities reaching up to 20 × 106 viable cells/mL with over 90 % viability 12 days post-inoculum, and antibody titers three times higher than corresponding batch cultures. By implementing a novel high-speed protein A fiber technology (Fibro PrismA) with a capture residence time of only 7.5 s, 8 L of supernatant could be purified in 4.5 h without compromising the purity, structural integrity and function of the bispecific antibody. Results from this study related to medium adaptation and design of fed-batch protocols will be highly beneficial during the forthcoming scale-up of this therapeutic antibody.

Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80–100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25–250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology.

KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3–4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.