Transfection, the process that enables the delivery of nucleic acids into cells to overexpress a protein of interest, and or to inhibit gene expression is an indispensable tool for the development of these novel strategies. With 20 years of expertise in transfection, Polyplus supports covid-19 research by providing innovative transfection reagents for gentle yet efficient delivery into cells.  

The covid-19 pandemic had an impact on research publishing. Science worldwide have put their specific expertise to contribute to a better understanding of SARS-CoV-2 genome sequence, structure, and mechanism of action to develop vaccines at unprecedented speed. While the first developed vaccines were administered less than a year after the beginning of the pandemic, the protection that these vaccines procure may be short-lived due to the emergence of new SARS-CoV-2 variants that evade vaccine immunity. Therefore, research is still actively strong to develop innovative strategies or alternatively repurpose existing treatments to fight against virus infection.  

Transfection, the process that enables the delivery of nucleic acids into cells to overexpress a protein of interest, and or to inhibit gene expression is an indispensable tool for the development of these novel strategies. With 20 years of expertise in transfection, Polyplus supports covid-19 research by providing innovative transfection reagents for gentle yet efficient delivery into cells.  


in vivo-jetRNA® to accelerate development of animal models for rapid evaluation of SARS-CoV-2 vaccination and treatments

Animal models are needed to better understand the adaptive immune response to SARS-CoV-2 and even more so for the testing of novel vaccines and antiviral strategies. Hassert and colleagues (2020) focused their research on developing such a mouse model. Their mouse model expresses the human ACE2 (hACE2) receptor to ensure that the interaction with the spike protein of SARS-CoV-2 leads to infection and immune response in mice. There are several strategies that have been tested to generate a mouse model expressing hACE2 receptor including CRISPR/Cas9 genome editing and viral vector-based strategies (AAV, adenovirus). Hassert et al. 2020 relied on a technology that makes generation of the mouse model easier and faster to push forward the development process of vaccines and treatment strategies, as well as provide a clear roadmap for other research teams. The winning combo: a non-viral directly injectable transfection reagent, in vivo-jetRNA® combined with mRNA. Using this approach, they could demonstrate that in vivo mRNA transfection with in vivo-jetRNA® led to i) cell surface expression of hACE2 receptor and ii) SARS-CoV-2 infection of in vivo transfected cells. 

Hassert et al. (2020) PLoS Pathog. mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2.  


FectoPRO® for scalable production of antibody against COVID-19 and novel strategies 

The coronavirus spike protein is key to virus entry into cells with its ability to bind at high affinity to the cell surface metallopreotease ACE2. Researchers are focusing on specific tertiary structures of the spike protein to develop potent treatments. To study the spike protein, the first step is recombinant protein production in mammalian cells. FectoPRO® transfection reagent is optimized for rapid and high-yield recombinant protein production in mammalian cell line and derivatives of HEK-293 and CHO cells grown in suspension. There are several derivatives of HEK-293 and CHO cell lines that have been developed to grow at higher density, and FectoPRO® can be utilized to transfect these higher cell density derivatives. Here is an example of novel strategies that could be beneficial as complementary treatments to vaccines, especially with the growing number of existing variants. In a recent preprint study, Grishin et al., 2021 used FectoPRO® to quickly produce in high cell density Expi293 cells the receptor binding domain (RBD) of the Spike protein and the corresponding domain of the ACE2 receptor. Their goal was to study whether disruption of the multiple disulfide bonds in the RBD of the SARS-CoV-2 Spike protein could disrupt its function, and therefore decrease its binding affinity to the ACE2 receptor. They demonstrated that reducing agents of disulfide bonds such as DTT and TCEP could have a strong antiviral effect and prevent infection.  

Grishin AM et al. (2021). bioRxiv. Spike protein disulfide disruption as a potential treatment for SARS-CoV-2. 


With the continuous surge of new SARS-Cov-2 variants due to multiple mutations in the spike protein, the question on everyone’s mind is how likely is it to get covid twice? Kaku and colleagues’ approach in understanding how these novel variants may change the rule of the game is by evaluating how well neutralizing antibodies against the alpha variant (B.1.1.7) can also neutralize existing variants beta (B.1.351) and gamma (P.1) – this is before the delta (B.1.617.2) variants. For this, plasma samples were recovered from two convalescent patients infected by the alpha variant in March 2020. To isolate SARS-CoV-2 neutralizing antibodies, IgG+ memory B cells from these patients were isolated and immunoglobulin variable genes were cloned to obtain IgG heavy and light chain expression plasmids. To overexpress these antibodies, heavy and light chain expression plasmids were co-transfected either in HEK-293T cells or ExpiCHO cells using respectively FectoPRO® transfection expression and FectoCHO® expression system. FectoCHO® expression system ensures high protein production yield in CHO cell lines (eg. CHO-S, CHO-K1, ExpiCHO-S). More than 400 antibodies isolated from each patient were transiently overexpressed, and out of these 30-50 antibodies were directed against the S protein, and only 1-4 antibodies had neutralizing activity against the alpha variant. The majority of the antibodies had a significant decreased neutralizing capacity against beta and gamma variants, which shows that it is important to monitor new variants that escape the immune system. 

Kaku Y et al. (2021) Cell Reports. Resistance of SARS-CoV-2 variants to neutralization by antibodies induced in convalescent patients with COVID-19 


Another piece to the puzzle is the development of a rapid, accurate and cost-effective serologic test to facilitate public health responses. Antibody testing is important to identify individuals that have developed protective immunity against SARS-CoV-2, as well as provide data on the duration of this immunity gained from previous exposure to the virus or thanks to the vaccination campaign. Esmail and colleagues, 2021 have developed such a test that is isotope independent unlike most existing antibody tests that are specific for an antibody (IgG, IgM, IgA). This test is based on an antibody-dependent agglutination assay on antigen-coated latex particles. Latex particles are surface coated with SARS-CoV-2 antigen, S-RBD or nucleocapsid and incubated with plasma. When containing the neutralizing antibodies, it induces agglutination of the latex particles. To coat the latex particles, proteins were transiently produced in high density Expi293F cells using FectoPRO® transfection reagent in a short amount of time, within a weeks’ time. 

Esmail S. et al., 2021 Cell Reports Methods. Rapid and accurate agglutination-based testing for SARS-CoV-2 antibodies 


jetOPTIMUS® a lab bench essential for coronavirus research

jetOPTIMUS® is a broad range DNA transfection reagent that significantly improves delivery in all adherent cell types, whether easy to transfect such as HEK-293 cells or hard to transfect cells such as primary mesenchymal stem cells. Adherent HEK-293 cells offer a great cell model to identify protein interactions that play a decisive role in viral infection. For example, Zhang et al., 2021 used jetOPTIMUS® to study intracellular protein-protein interactions between NSP1 protein of SARS-CoV-2 and the mRNA export receptor NXF1 NXT1. Alike influenza viruses, NSP1 protein may also block endogenous gene expression in favor of its own replication by targeting the mRNA nuclear export machinery. jetOPTIMUS® was used to transfect adherent HEK-293 cells with plasmid DNA encoding for different constructs of NSP1 to perform GST pulldown and co-immunoprecipitation of NXF1 NXT1 from cell lysates. 

Zhang K. et al. (2021) Sci Adv 7, eabe7386. Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression. 


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