Production of trimeric SARS-CoV-2 spike protein by CHO cells for serological COVID-19 testing.

We describe scalable and cost-efficient production of full length, His-tagged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme-linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID-19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.

Molecular design of controllable recombinant adeno-associated virus (AAV) expression systems for enhanced vector production.

Recombinant adeno-associated virus (rAAV) is the leading vector for the delivery of gene therapies. However, low viral genome (VG) titers are common and the proportion of "full" capsids containing the therapeutic gene payload can be highly variable. The coordinated molecular design of plasmids encoding viral components and Helper functions remains a major challenge for rAAV manufacturing. Here we present the design of improved Rep/Cap and Helper plasmids for rAAV2/8 production, (i) a Rep/Cap expression vector harboring independently controllable rep and cap genes and (ii) an improved Helper plasmid harboring E4 gene deletion variants. First, an optimized Rep/Cap vector utilized a truncated p5 promoter, a p5 cis-regulatory element at the 3' end in combination with a heterologous promoter to drive Cap expression and an additional copy of the rep52/40 gene to overexpress short Rep proteins. We demonstrate that Rep78 is essential for efficient rAAV2/8 production in HEK293 cells, and a higher ratio of short Rep to long Rep proteins enhances genome packaging. Second, we identified regulators and open reading frames within the Helper plasmid that contribute to increased rAAV2/8 production. While L4-33k/22k is integral to optimal production, the use of E4orf6-6/7 subset significantly enhanced VG titer. Together, an optimal combination of engineered Rep/Cap and Helper plasmid variants increased VG titer by 3.1-fold. This study demonstrates that configuring and controlling the expression of the different AAV genetic elements contributes toward high rAAV production and product quality (full/empty capsid ratio).

Increased recombinant adeno-associated virus production by HEK293 cells using small molecule chemical additives.

Recombinant adeno-associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile allowing broad clinical application. Recent successes in rAAV-mediated gene therapy clinical trials will continue to drive demand for improved rAAV production processes to reduce costs. Here, we demonstrate that small molecule bioactive chemical additives can significantly increase recombinant AAV vector production by human embryonic kidney (HEK) cells up to three-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective histone deacetylase inhibitor) were identified as positive regulators of rAAV8 genome titre in a microplate screening assay. Addition of nocodazole to triple-transfected HEK293 suspension cells producing rAAV arrested cells in G2/M phase, increased average cell volume and reduced viable cell density relative to untreated rAAV producing cells at harvest. Final crude genome vector titre from nocodazole treated cultures was >2-fold higher compared to non-treated cultures. Further investigation showed nocodazole addition to cultures to be time critical. Genome titre improvement was found to be scalable and serotype independent across two distinct rAAV serotypes, rAAV8 and rAAV9. Furthermore, a combination of M344 and nocodazole produced a positive additive effect on rAAV8 genome titre, resulting in a three-fold increase in genome titre compared to untreated cells.

Engineering of the CMV promoter for controlled expression of recombinant genes in HEK293 cells.

Expression of recombinant genes in HEK293 cells is frequently utilized for production of recombinant proteins and viral vectors. These systems frequently employ the cytomegalovirus (CMV) promoter to drive recombinant gene transcription. However, the mechanistic basis of CMV-mediated transcriptional activation in HEK293 cells is unknown and consequently there are no strategies to engineer CMV for controlled expression of recombinant genes. Extensive bioinformatic analyses of transcription factor regulatory elements (TFREs) within the human CMV sequence and transcription factor mRNAs within the HEK293 transcriptome revealed 80 possible regulatory interactions. Through in vitro functional testing using reporter constructs harboring discrete TFREs or CMV deletion variants we identified key TFRE components and clusters of TFREs (cis-regulatory modules) within the CMV sequence. Our data reveal that CMV activity in HEK293 cells is a function of the promoters various constituent TFREs including AhR:ARNT, CREB, E4F, Sp1, ZBED1, JunB, c-Rel, and NF-κB. We also identified critical Sp1-dependent upstream activator elements near the transcriptional start site that were required for efficient transcription and YY1 and RBP-Jκ binding sites that mediate transrepression. Our study shows for the first time that novel, compact CMV-derived promoters can be engineered that exhibit up to 50% higher transcriptional efficiency (activity per unit DNA sequence) or 14% increase in total activity compared to the wild-type counterpart.