Heterogeneity of acetylcholine receptor autoantibody-mediated complement activity in patients with myasthenia gravis.

Background: Autoantibodies targeting the acetylcholine receptor (AChR) in the serum of myasthenia gravis (MG) patients are broadly polyclonal and heterogeneous in their pathogenic capacity. Specifically, AChR autoantibody-mediated pathology occurs through three mechanisms that include complement-directed tissue damage, blocking of the acetylcholine binding site on the AChR, and modulation (internalization) of the AChR. Clinical assays used for diagnosis and prognosis measure only AChR autoantibody binding and they provide weak association with disease burden, thereby limiting understanding of mechanistic heterogeneity, and monitoring therapeutic response. Objective: To develop an in-vitro cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation. Methods: A HEK293T cell line, which is commonly used for live cell-based AChR autoantibody binding assays, was modified such that the expression of the complement regulator genes (CD46, CD55 and CD59) were disrupted using CRISPR/Cas9 genome editing. This modified cell line was used to measure serum AChR autoantibody-mediated complement MAC formation via flow cytometry. Results: AChR autoantibody-mediated MAC formation required the use of a modified HEK293T cell line in which the surface expression of three complement regulator genes was absent. Serum samples (n=155) from 97 clinically confirmed AChR patients were tested along with 32 healthy donor (HD) samples; the MG cohort included a wide range of disease burden and AChR autoantibody titer. AChR autoantibodies were detected in 139 of the 155 (89.7%) AChR patient samples via a live cell-based assay. Of the 139 AChR positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), while no autoantibodies or MAC formation was detected in samples from the HD group. Autoantibody-mediated MAC formation positively associated with autoantibody binding in most MG patient samples. However, a subset displayed a disassociation between binding and MAC formation. Conclusions: We demonstrate the development of a novel assay for evaluating AChR autoantibody-mediated complement activity. It is anticipated that this assay will afford a deeper understanding of the heterogeneous disease pathology and allow for the identification of MG patients who may benefit from complement inhibitor therapy.

Generation and utilization of a HEK-293T murine GM-CSF expressing cell line

Macrophages and dendritic cells (DCs) are innate immune cells that play a key role in defense against pathogens. In vitro cultures of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) are well-established and valuable methods for immunological studies. Typically, commercially available recombinant GM-CSF is utilized to generate BMDCs and is also used to culture alveolar macrophages. We have generated a new HEK-293T cell line expressing murine GM-CSF that secretes high levels of GM-CSF (~180 ng/ml) into complete media as an alternative to commercial GM-CSF. Differentiation of dendritic cells and expression of various markers were kinetically assessed using the GM-CSF HEK293T cell line, termed supGM-CSF and compared directly to purified commercial GMCSF. After 7–9 days of cell culture the supGM-CSF yielded twice as many viable cells compared to the commercial purified GM-CSF. In addition to differentiating BMDCs, the supGM-CSF can be utilized to culture functionally active alveolar macrophages. Collectively, our results show that supernatant from our GM-CSF HEK293T cell line supports the differentiation of mouse BMDCs or alveolar macrophage culturing, providing an economical alternative to purified GM-CSF.

Cell Banking of HEK293T cell line for clinical-grade lentiviral particles manufacturing

Background Cell banks are widely used to preserve cell properties as well as to record and control the use of cell lines in biomedical research. The generation of cell banks for the manufacturing of Advanced Therapy Medicinal Products, such as cell and gene therapy products, must comply with current Good Manufacturing Practice regulations. The quality of the cell lines used as starting materials in viral-vector manufacturing processes must be also assessed. Methods Three batches of a Master Cell Bank and a Working Cell Bank of the HEK293T cell line were manufactured under current Good Manufacturing Practices regulations. Quality control tests were performed according to product specifications. Process validation includes the training of manufacturing personnel by performing simulation tests, and the continuous measurement of environmental parameters such as air particles and microorganisms. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products. Results All batches of HEK293T Master and Working Cell Banks met the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working Cell Banks maintained the defined cell viability and concentration over a 37 month-period after cryopreservation. Conclusions Manufacturing cell banks under Good Manufacturing Practice regulations for their use as raw materials or final cellular products is feasible. HEK293T cell banks were used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

Cell Banking of HEK293T cell line for clinical-grade lentiviral particles manufacturing

Background: Cell banks are widely used to preserve cell properties as well as to record and control the use of cell lines in biomedical research. The generation of cell banks for the manufacturing of Advanced Therapy Medicinal Products, such as cell and gene therapy products, must comply with current Good Manufacturing Practice regulations. The quality of the cell lines used as starting materials in viral-vector manufacturing processes must be also assessed.Methods: Three batches of a Master Cell Bank and a Working Cell Bank of the HEK293T cell line were manufactured under current Good Manufacturing Practices regulations. Quality control tests were performed according to product specifications. Process validation includes the training of manufacturing personnel by performing simulation tests, and the continuous measurement of environmental parameters such as air particles and microorganisms. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of HEK293T Master and Working Cell Banks met the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working Cell Banks maintained the defined cell viability and concentration over a 37 month-period after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practice regulations for their use as raw materials or final cellular products is feasible. HEK293T cell banks were used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

Cell Banking of HEK293T Cell Line for Clinical-Grade Lentiviral Particles Manufacturing

Background: Cell banks have been widely used to preserve cell properties as well as to record and control cell line access in research. However, the generation of cell banks involved in the manufacturing of Advanced therapy medicinal products such as cell or gene therapy products must comply with the current Good Manufacturing Practice regulation. Similarly, the quality of those cell lines used as starting materials in viral-vector manufacturing processes must be also evaluated.Methods: Three batches of both Master Cell Bank and Working Cell Bank of the HEK293T cell line were manufactured under the current Good Manufacturing Practices regulation. Quality control test were performed according to the product specifications. The process validation includes previous qualification of the manufacturing personnel by performing simulation tests as well as the continuous measure of environmental parameters during manufacturing such as air particles and microorganism. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of Master Cell Bank and Working Cell Bank fulfilled the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working cell bank maintain the defined viability and cell number 37 months after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practices regulation for its use as raw material or final cellular product is feasible. HEK293T cell banks have been used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

Cell Banking of HEK293T Cell Line for Clinical-Grade Lentiviral Particles Manufacturing

Background: Cell banks have been widely used to preserve cell properties as well as to record and control cell line access in research. However, the generation of cell banks involved in the manufacturing of Advanced therapy medicinal products such as cell or gene therapy products must comply with the current Good Manufacturing Practice regulation. Similarly, the quality of those cell lines used as starting materials in viral-vector manufacturing processes must be also evaluated.Methods: Three batches of both Master Cell Bank and Working Cell Bank of the HEK293T cell line were manufactured under the current Good Manufacturing Practices regulation. Quality control test were performed according to the product specifications. The process validation includes previous qualification of the manufacturing personnel by performing simulation tests as well as the continuous measure of environmental parameters during manufacturing such as air particles and microorganism. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of Master Cell Bank and Working Cell Bank fulfilled the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working cell bank maintain the defined viability and cell number 37 months after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practices regulation for its use as raw material or final cellular product is feasible. HEK293T cell banks have been used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

Generation and utilization of a HEK-293T murine GM-CSF expressing cell line

Macrophages and dendritic cells (DCs) are innate immune cells that play a key role in defense against pathogens. In vitro cultures of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) are well-established and valuable methods for immunological studies. Typically, commercially available recombinant GMCSF is utilized to generate BMDCs and is also used to culture alveolar macrophages. We have generated a new HEK-293T cell line expressing murine GM-CSF that secretes high levels of GM-CSF (∼180ng/ml) into complete media as an alternative to commercial GM-CSF. Differentiation of dendritic cells and expression of various markers were kinetically assessed using the GM-CSF HEK293T cell line, termed supGM-CSF and compared directly to purified commercial GMCSF. After 7-9 days of cell culture the supGM-CSF yielded twice as many viable cells compared to the commercial purified GM-CSF. In addition to differentiating BMDCs, the supGM-CSF can be utilized to culture alveolar macrophages without an altering inflammatory activation cascade. Collectively, our results show that supernatant from our GM-CSF HEK293T cell line supports the differentiation of mouse BMDCs or alveolar macrophage culturing, providing an economical alternative to purified GM-CSF.

Enhance genome editing efficiency and specificity by a quick CRISPR/Cas9 system

CRISPR/Cas9 is a powerful genome editing tool that has been successfully applied to a variety of species, including zebrafish. However, targeting efficiencies vary greatly at different genomic loci, the underlying causes of which were still elusive. Here we report a quick CRISPR/Cas9 system, designated as qCas9, which exhibits accelerated turnover of Cas9 protein in zebrafish. Our data showed that qCas9 significantly improved targeting efficiency, including both knock-out and knock-in in F0 embryos, and yielded higher germline transmission rate in founder screen. Importantly, qCas9 showed little to no off-target editing in zebrafish and profoundly reduced off-target effect in HEK293T cell line. In summary, our findings demonstrate that qCas9 is a simple, economic and highly effective method to improve genome editing efficiency in zebrafish embryos and also holds great potential in reducing off-target effect in mammalian cell lines.