Chinese Hamster Ovary Research Articles

Technoeconomic Analysis of Intensified PEGylated Biopharmaceutical Recombinant Protein Production: Alpha Antitrypsin as a Model Case

Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder characterized by the insufficient production of the AAT protein. Due to availability limitations, not all AATD patients receive protein therapy treatment. In this study, the technoeconomic analysis of different processes (conventional and intensified) producing 200 kg/year of PEGylated recombinant AAT (PEG-AAT) using a Chinese hamster ovary cell line was investigated. All bioprocesses consist of upstream, downstream, and PEGylation sections. A base-case model (process A) of the conventional fed-batch production bioreactor was developed using SuperPro Designer software (Version 13) to evaluate the economic feasibility of the process. The cost of goods (COG) was estimated to be approximately USD 387.6/g. Furthermore, an intensified process (B) was modeled and evaluated to reduce the COG. Process intensification was implemented in the process (N-1 perfusion bioreactor). The specific operating COG for process B was found to be 10% less than that of process A. Scenario analysis was performed to assess the impact of process capacity (100–1000 kg/year) and cell-specific productivity (30–90 pg/cell/day). With an increase in process capacity, the specific operating COG was reduced for all processes. Increasing cell-specific productivity decreases the specific operating COG at different rates for each process, depending on the titer level. Future investigations into the PEGylation section are required since it has the highest COG of all the sections.

Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10–15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1–2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

SPEED-MODE cell line development (CLD): Reducing Chinese hamster ovary (CHO) CLD timelines via earlier suspension adaptation and maximizing time spent in the exponential growth phase.

Chinese hamster ovary (CHO) cells are the preferred system for expression of therapeutic proteins and the majority of all biotherapeutics are being expressed by these cell lines. CHO expression systems are readily scalable, resistant to human adventitious agents, and have desirable post-translational modifications, such as glycosylation. Regardless, drug development as a whole is a very costly, complicated, and time-consuming process. Therefore, any improvements that result in reducing timelines are valuable and can provide patients with life-saving drugs earlier. Here we report an effective method (termed SPEED-MODE, herein) to speed up the Cell line Development (CLD) process in a targeted integration (TI) CHO CLD system. Our findings show that (1) earlier single cell cloning (SCC) of transfection pools, (2) speeding up initial titer screening turnaround time, (3) starting suspension adaptation of cultures sooner, and (4) maximizing the time CHO cultures spend in the exponential growth phase can reduce CLD timelines from ~4 to ~3 months. Interestingly, SPEED-MODE timelines closely match the theoretical minimum timeline for CHO CLD assuming that CHO cell division is the rate limiting factor. Clones obtained from SPEED-MODE CLD yielded comparable titer and product quality to those obtained via a standard CLD process. Hence, SPEED-MODE CLD is advantageous for manufacturing biotherapeutics in an industrial setting as it can significantly reduce CLD timelines without compromising titer or product quality.

Boltzmann Model Predicts Glycan Structures from Lectin Binding.

Glycans are complex oligosaccharides that are involved in many diseases and biological processes. Unfortunately, current methods for determining glycan composition and structure (glycan sequencing) are laborious and require a high level of expertise. Here, we assess the feasibility of sequencing glycans based on their lectin binding fingerprints. By training a Boltzmann model on lectin binding data, we predict the approximate structures of 88 ± 7% of N-glycans and 87 ± 13% of O-glycans in our test set. We show that our model generalizes well to the pharmaceutically relevant case of Chinese hamster ovary (CHO) cell glycans. We also analyze the motif specificity of a wide array of lectins and identify the most and least predictive lectins and glycan features. These results could help streamline glycoprotein research and be of use to anyone using lectins for glycobiology.

Effect of 4-phenylbutyrate addition timing on titer of Fc-fusion protein in Chinese hamster ovary cell cultures

Effect of 4-phenylbutyrate addition timing on titer of Fc-fusion protein in Chinese hamster ovary cell cultures

Conserved cysteines prevent C-mannosylation of mucin Cys domains.

Mucins are major components of the mucus. Besides the highly O-glycosylated tandem repeat domains, mucins contain Cys domains (CysDs). CysDs contain conserved disulfide-forming cysteine residues as well as a WxxW motif. Since this is the consensus sequence for tryptophan C-mannosylation, mucin CysDs have been suggested to be targets for C-mannosyltransferases, but this has never been directly shown. Here, we recombinantly expressed human mucinCysDs in Chinese hamster ovary (CHO) cells and analyzed the C-mannosylation status. Mass spectrometric analysis revealed that the putative C-mannose site is not or only barely C-mannosylated. However, mutation of the adjacent cysteine residues enabled C-mannosylation to occur. In contrast to mucin CysDs, the homologous CysD of human cartilage intermediate layer protein 1 (CILP1) lacks these cysteine residues preceding the WxxW motif. We show that CILP1 CysD is C-mannosylated, but introducing a cysteine at the -2 position causes this modification to be lost. We thus conclude that the presence of cysteine residues prevents the modification of the WxxW motif in CysDs.

Mammalian Cell Genotoxicity of Potable Reuse and Conventional Drinking Waters.

Potable reuse water is increasingly part of the water supply portfolio for municipalities facing water shortages, and toxicity assays can be useful for evaluating potable reuse water quality. We examined the Chinese hamster ovary cell acute direct genotoxicity of potable reuse waters contributed by disinfection byproducts (DBPs) and anthropogenic contaminants and used the local conventional drinking waters as benchmarks for evaluating potable reuse water quality. Our results showed that treatment trains based on reverse osmosis (RO) were more effective than RO-free treatment trains for reducing the genotoxicity of influent wastewaters. RO-treated reuse waters were less genotoxic than the local tap water derived from surface water, whereas reuse waters not treated by RO were similarly genotoxic as the local drinking waters when frequent replacement of granular activated carbon limited contaminant breakthrough. The genotoxicity contributed by nonvolatile, uncharacterized DBPs and anthropogenic contaminants accounted for ≥73% of the total genotoxicity. The (semi)volatile DBPs of current research interest contributed 2-27% toward the total genotoxicity, with unregulated DBPs being more important genotoxicity drivers than regulated DBPs. Our results underscore the need to look beyond known, (semi)volatile DBPs and the importance of determining whole water toxicity when assessing the quality of disinfected waters.

Use of spectroscopic process analytical technology for rapid quality evaluation during preparation of CHO cell culture media.

Media preparation parameters contribute significantly to media quality, cell culture performance, productivity, and product quality. Establishing proper media preparation procedures is critical for ensuring a robust CHO cell culture process. Process analytical technology (PAT) enables unique ways to quantify assessments and improve media quality. Here, cell culture media were prepared under a wide range of temperatures (40-80°C) and pH (7.6-10.0). Media quality profiles were compared using three real-time PATs: Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, and excitation-emission matrix (EEM) spectroscopy. FTIR and Raman spectroscopies identified shifts in media quality under high preparation temperature (80°C) and at differing preparation pH which negatively impacted monoclonal antibody (mAb) production. In fed-batch processes for production of three different mAbs, viable cell density (VCD) and cell viability were mostly unaffected under all media preparation temperatures, while titer and cell specific productivity of mAb decreased when cultured in basal and feed media prepared at 80°C. High feed preparation pH alone was tolerated but cell growth and productivity profiles deviated from the control condition. Further, charge variants (main, acidic, basic species) and glycosylation (G0F, afucosylation, and high mannose) were examined. Statistically significant differences were observed for one or more of these quality attributes with any shifts in media preparation. In this study, we demonstrated strong associations between media preparation conditions and cell growth, productivity, and product quality. The rapid evaluation of media by PAT implementation enabled more comprehensive understanding of different parameters on media quality and consequential effects on CHO cell culture.

Evaluation of the Genotoxicity of Almond Hull: Implications for Its Use as a Novel Food Ingredient.

Almond hull, a substantial byproduct comprising more than half of almond fresh weight, has recently gained attention due to its functionality and sustainability benefits. Despite heightened interest, information regarding its toxicity remains limited. In order to assess its genotoxic potential, we conducted Good Laboratory Practice-compliant in vitro and in vivo studies following Organization for Economic Co-operation and Development (OECD) guidelines. No evidence of toxicity or mutagenicity was observed in a bacterial reverse mutation assay using five tester strains, evaluating almond hull at concentrations up to 5 mg/plate, with or without metabolic activation. Almond hull did not induce chromosome structural damage in a chromosome aberration assay using Chinese hamster ovary cells, nor did it cause any spermatogonial chromosomal aberration in tested male BALB/c mice. To evaluate its ability to induce DNA damage in rodents, a combined micronucleus assay was conducted in KM mice of both sexes. Almond hull was administered at doses of 1250, 2500, and 5000 mg/kg/day via gavage once daily for 2 days. No adverse effects of almond hull were observed in the micronucleus assay. Our results indicate no evidence of the genotoxic potential of almond hull administered up to the maximum concentrations of 5 g/kg, as recommended by OECD guidelines.

Abstract PO5-18-06: On-going phase 1A clinical trial of A01, a chimerized monoclonal antibody to Progranulin/Glycoprotein 88 (GP88) in patients with advanced malignancy

Abstract Progranulin (PGRN/GP88) is an 88 kDa glycoprotein characterized by seven and a half double cysteine rich repeats which is the largest member of the granulin-epithelin protein family. PGRN/GP88 has been demonstrated as a biological driver of tumorigenesis, survival, and drug resistance in several cancers including breast cancer (BC), lung prostate, ovarian and digestive cancers. PGRN/GP88 tissue expression is an independent prognostic factor of recurrence in breast, lung cancers while elevated serum PGRN/GP88 level in metastatic breast, lung, ovarian and prostate cancer patients. Elevated PGRN/GP88 levels are associated with poor outcomes such as progression and shortened survival. An anti-human PGRN/GP88 monoclonal antibody able to inhibit the the in vitro and in vivo action of human PGRN/GP88 has been developed, chimerized and expressed in CHO cells. All IND enabling activities including pharmacology, GMP manufacturing, formulation and GLP toxicology studies have been conducted. The IND application has been filed and cleared by the Food and Drug Administration. A first-in-human, first-in-class phase 1 safety and efficacy clinical study of AG01 in patients with solid tumors and advanced disease with special focus on patients with breast, lung and ovarian cancers has been initiated and is on-going at the University of Maryland Greenebaum Cancer Center. The trial is registered as NCT05627960 to clinicaltrials.gov site. The presentation will provide an update on the number of patients enrolled in this on-going phase 1A trial. Supported by grants R44CA162629 and R44CA224718 from the National Cancer Institute to GS. Citation Format: Katherine Tkaczuk, Paula Rosenblatt, Ranee Mehra, Katherine Scilla, Nancy Tait, Binbin Yue, Ginette Serrero. On-going phase 1A clinical trial of A01, a chimerized monoclonal antibody to Progranulin/Glycoprotein 88 (GP88) in patients with advanced malignancy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-18-06.

Safety and immunogenicity of the SARS-CoV-2 LYB001 RBD-based VLP vaccine (CHO cell) phase 1 in Chinese adults: a randomized, double-blind, positive-parallel-controlled study

ABSTRACT Background Vaccination remains the cornerstone of defense against COVID-19 globally. This study aims to assess the safety and immunogenicity profile of innovative vaccines LYB001. Research design and methods This was a randomized, double-blind, parallel-controlled trial, in 100 healthy Chinese adults (21 to 72 years old). Three doses of 30 or 60 µg of SARS-CoV-2 RBD-based VLP vaccine (LYB001), or the SARS-CoV-2 RBD-based protein subunit vaccine (ZF2001, control group) were administered with a 28-day interval. Differences in the incidence of adverse events (AEs) and indicators of humoral and cellular immunity among the different groups were measured. Results No severe adverse events were confirmed to be vaccine-related, and there was no significant difference in the rate of adverse events between the LYB001 and control group or the age subgroups (p > 0.05). The LYB001 groups had significantly higher or comparable levels of seroconversion rates, neutralization antibody, S protein-binding antibody, and cellular immunity after whole vaccination than the control group. Conclusions Our findings support that LYB001 developed on the VLP platform is safe and well tolerated with favorable immunogenicity for fundamental vaccination in healthy adults. Therefore, further larger-scale clinical studies are warranted. Trial Registration This trial was registered with ClinicalTrials.gov (NCT05552573).

Recombinant human fibroblast growth factor 7 obtained from stable Chinese hamster ovary cells enhances wound healing.

Although fibroblast growth factor 7 (FGF7) is known to promote wound healing, its mass production poses several challenges and very few studies have assessed the feasibility of producing FGF7 in cell lines such as Chinese hamster ovary (CHO) cells. Therefore, this study sought to produce recombinant FGF7 in large quantities and evaluate its wound healing effect. To this end, the FGF7 gene was transfected into CHO cells and FGF7 production was optimized. The wound healing efficacy of N-glycosylated FGF7 was evaluated in animals on days 7 and 14 post-treatment using collagen patches (CPs), FGF7-only, and CP with FGF7 (CP+FGF7), whereas an untreated group was used as the control. Wound healing was most effective in the CP+FGF7 group. Particularly, on day 7 post-exposure, the CP+FGF7 and FGF7-only groups exhibited the highest expression of hydroxyproline, fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor. Epidermalization in H&E staining showed the same order of healing as hydroxyproline content. Additionally, the CP+FGF7 and FGF7-only group exhibited more notable blood vessel formation on days 7 and 14. In conclusion, the prepared FGF7 was effective in promoting wound healing and CHO cells can be a reliable platform for the mass production of FGF7.

Nature as blueprint: Global phenotype engineering of CHO production cells based on a multi-omics comparison with plasma cells

Especially for the production of artificial, difficult to express molecules a further development of the CHO production cell line is required to keep pace with the continuously increasing demands. However, the identification of novel targets for cell line engineering to improve CHO cells is a time and cost intensive process. Since plasma cells are evolutionary optimized for a high antibody expression in mammals, we performed a comprehensive multi-omics comparison between CHO and plasma cells to exploit optimized cellular production traits. Comparing the transcriptome, proteome, miRNome, surfaceome and secretome of both cell lines identified key differences including 392 potential overexpression targets for CHO cell engineering categorized in 15 functional classes like transcription factors, protein processing or secretory pathway. In addition, 3 protein classes including 209 potential knock-down/out targets for CHO engineering were determined likely to affect aggregation or proteolysis. For production phenotype engineering, several of these novel targets were successfully applied to transient and transposase mediated overexpression or knock-down strategies to efficiently improve productivity of CHO cells. Thus, substantial improvement of CHO productivity was achieved by taking nature as a blueprint for cell line engineering.

Enhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures.

The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields. In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines. Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.

Glycosylation Modulation Dictates Trafficking and Interaction of SARS-CoV-2 S1 Subunit and ACE2 in Intestinal Epithelial Caco-2 Cells

Glycosylation Modulation Dictates Trafficking and Interaction of SARS-CoV-2 S1 Subunit and ACE2 in Intestinal Epithelial Caco-2 Cells

Disinfection byproducts and their cytotoxicity contribution from dissolved black carbon in source water during chlor(am)ination

Dissolved black carbon (DBC), the soluble component of black carbon, which mainly comes from the incomplete combustion of fossil fuels or biomass, is widely spread in source water and significantly contributes to the formation of dissolved organic matter (DOM). However, the origin of DBC in different types of source water in China has not been well studied, as well as its subsequent transformation and toxicity contribution during disinfection of source water DOM by chlor(am)ine. In this study, DBC from 17 different source water in East China at different seasons was collected. The δ13C compositions indicated that straw burning was the main origin of DBC in source water. After simulated chlor(am)ination of DBC, 5 categories of aliphatic disinfection byproducts (DBPs) including trihalomethanes, haloacetic acids, haloacetonitriles, haloketones, halonitromethanes and 6 categories of aromatic DBPs including halophenols, halonitrophenols, halohydroxybenzaldehyde, halohydroxybenzoic acid, halobenzoquinones and haloaniline were detected. Compared with chlorination of DBC, higher levels of nitrogenous DBPs and aromatic DBPs were generated during chloramination. Detected DBPs accounted for 42 % of total organic halogen. What's more, Chinese hamster ovary cells cytotoxicity tests showed that the cytotoxicity of DBPs formed by chlor(am)ination of DBC was 4 times higher than that by chlor(am)ination of DOM. Haloacetonitriles contributed to the highest cytotoxicity in the chloramination of DBC, and haloacetic acids contributed to the highest cytotoxicity in chlorination. 67 % of the total cytotoxicity attributed to the undetected DBPs. As a result, DBPs generated from DBC contributed to 11.7 % of the total cytotoxicity in the chlor(am)ination of the source water DOM although DBC only took up 2 % of DOC in the source water. Results obtained from this study systematically revealed the DBPs formation from DBC and their potential cytotoxicity contribution in the chlor(am)ination of source water DOM, which should not be ignored in drinking water treatment.

Machine Learning and Deep Learning Strategies for Chinese Hamster Ovary Cell Bioprocess Optimization

The use of machine learning and deep learning has become prominent within various fields of bioprocessing for countless modeling and prediction tasks. Previous reviews have emphasized machine learning applications in various fields of bioprocessing, including biomanufacturing. This comprehensive review highlights many of the different machine learning and multivariate analysis techniques that have been utilized within Chinese hamster ovary cell biomanufacturing, specifically due to their rising significance in the industry. Applications of machine and deep learning within other bioprocessing industries are also briefly discussed.

Tailored multivalent peptide targeting the B-subunit pentamer of cholera toxin inhibits its intestinal toxicity by inducing aberrant transport of the toxin in cells

Cholera toxin (Ctx) is a major virulence factor produced by Vibrio cholerae that can cause gastrointestinal diseases, including severe watery diarrhea and dehydration, in humans. Ctx binds to target cells through multivalent interactions between its B-subunit pentamer and the receptor ganglioside GM1 present on the cell surface. Here, we identified a series of tetravalent peptides that specifically bind to the receptor-binding region of the B-subunit pentamer using affinity-based screening of multivalent random-peptide libraries. These tetravalent peptides efficiently inhibited not only the cell-elongation phenotype but also the elevated cAMP levels, both of which are induced by Ctx treatment in CHO cells or a human colon carcinoma cell line (Caco-2 cells), respectively. Importantly, one of these peptides, NRR-tet, which was highly efficient in these two activities, markedly inhibited fluid accumulation in the mouse ileum caused by the direct injection of Ctx. In consistent, NRR-tet reduced the extensive Ctx-induced damage of the intestinal villi. After NRR-tet bound to Ctx, the complex was incorporated into the cultured epithelial cells and accumulated in the recycling endosome, affecting the retrograde transport of Ctx from the endosome to the Golgi, which is an essential process for Ctx to exert its toxicity in cells. Thus, NRR-tet may be a novel type of therapeutic agent against cholera, which induces the aberrant transport of Ctx in the intestinal epithelial cells, detoxifying the toxin.

Cholestane-3β,5α,6β-Triol Inhibits Acid-Sensing Ion Channels and Reduces Acidosis-Mediated Ischemic Brain Injury.

Activation of the acid-sensing ion channels (ASICs) by tissue acidosis, a common feature of brain ischemia, contributes to ischemic brain injury, while blockade of ASICs results in protection. Cholestane-3β,5α,6β-triol (Triol), a major cholesterol metabolite, has been demonstrated as an endogenous neuroprotectant; however, the mechanism underlying its neuroprotective activity remains elusive. In this study, we tested the hypothesis that inhibition of ASICs is a potential mechanism. The whole-cell patch-clamp technique was used to examine the effect of Triol on ASICs heterogeneously expressed in Chinese hamster ovary cells and ASICs endogenously expressed in primary cultured mouse cortical neurons. Acid-induced injury of cultured mouse cortical neurons and middle cerebral artery occlusion-induced ischemic brain injury in wild-type and ASIC1 and ASIC2 knockout mice were studied to examine the protective effect of Triol. Triol inhibits ASICs in a subunit-dependent manner. In Chinese hamster ovary cells, it inhibits homomeric ASIC1a and ASIC3 without affecting ASIC1β and ASIC2a. In cultured mouse cortical neurons, it inhibits homomeric ASIC1a and heteromeric ASIC1a-containing channels. The inhibition is use-dependent but voltage- and pH-independent. Structure-activity relationship analysis suggests that hydroxyls at the 5 and 6 positions of the A/B ring are critical functional groups. Triol alleviates acidosis-mediated injury of cultured mouse cortical neurons and protects against middle cerebral artery occlusion-induced brain injury in an ASIC1a-dependent manner. Our study identifies Triol as a novel ASIC inhibitor, which may serve as a new pharmacological tool for studying ASICs and may also be developed as a potential drug for treating stroke.

Variable Penetrance and Expressivity of a Rare Pore Loss-of-Function Mutation (p.L889V) of Nav1.5 Channels in Three Spanish Families.

A novel rare mutation in the pore region of Nav1.5 channels (p.L889V) has been found in three unrelated Spanish families that produces quite diverse phenotypic manifestations (Brugada syndrome, conduction disease, dilated cardiomyopathy, sinus node dysfunction, etc.) with variable penetrance among families. We clinically characterized the carriers and recorded the Na+ current (INa) generated by p.L889V and native (WT) Nav1.5 channels, alone or in combination, to obtain further insight into the genotypic-phenotypic relationships in patients carrying SCN5A mutations and in the molecular determinants of the Nav1.5 channel function. The variant produced a strong dominant negative effect (DNE) since the peak INa generated by p.L889V channels expressed in Chinese hamster ovary cells, either alone (-69.4 ± 9.0 pA/pF) or in combination with WT (-62.2 ± 14.6 pA/pF), was significantly (n ≥ 17, p < 0.05) reduced compared to that generated by WT channels alone (-199.1 ± 44.1 pA/pF). The mutation shifted the voltage dependence of channel activation and inactivation to depolarized potentials, did not modify the density of the late component of INa, slightly decreased the peak window current, accelerated the recovery from fast and slow inactivation, and slowed the induction kinetics of slow inactivation, decreasing the fraction of channels entering this inactivated state. The membrane expression of p.L889V channels was low, and in silico molecular experiments demonstrated profound alterations in the disposition of the pore region of the mutated channels. Despite the mutation producing a marked DNE and reduction in the INa and being located in a critical domain of the channel, its penetrance and expressivity are quite variable among the carriers. Our results reinforce the argument that the incomplete penetrance and phenotypic variability of SCN5A loss-of-function mutations are the result of a combination of multiple factors, making it difficult to predict their expressivity in the carriers despite the combination of clinical, genetic, and functional studies.