plasmid encoding ace 2  (Addgene inc)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Choice of cell line for sACE2 2 .v2.4-IgG1 production impacts pharmacokinetics and glycosylation (A and B) sACE2 2 .v2.4-IgG1 was expressed and purified from human Expi293F (gray) or nonhuman ExpiCHO-S (black) cultures that were both transiently transfected. A 10 mg/kg amount of sACE2 2 .v2.4-IgG1 was injected into the tail vein of human FcRn transgenic mice. (A) ACE2 catalytic activity and (B) protein concentrations based on ELISA were measured in plasma. Data are mean ± SEM, n = 3 mice per time point. (C) N-glycan types from glycomics analysis of sACE2 2 .v2.4-IgG1 produced in ExpiCHO-S vs. Expi293F cells. (D) Abundance of sialylated (Neu5Ac) and fucosylated N-glycan structures. (E and F) O-Glycan analysis of sACE2 2 .v2.4-IgG1 produced in (E) ExpiCHO-S and (F) Expi293F cells. After PNGaseF treatment of protein, O-glycans were released, purified, permethylated, and analyzed by MALDI-TOF-MS. O-glycan structures were assigned using Glycoworkbench software based on precursor masses and the common mammalian biosynthetic pathway. Glycan structures are indicated on the x axis by their m/z ratio. GlcNAc, blue squares; GalNAc, yellow squares; Gal, yellow circles; Neu5Ac, purple diamonds.

Article Snippet: For measuring kinetics of sACE2 2 -IgG1/RBD interactions, responses were recorded on an Octet RED96a and analyzed with a 1:1 binding model (global fit) using instrument software (Sartorius). sACE2 2 -IgG1 proteins were immobilized at 100 nM for 60 s to AHC (anti-human IgG Fc) biosensors (Sartorius) in assay buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20, 0.5% non-fat dry milk).

Techniques: Purification, Transfection, Injection, Transgenic Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Produced, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: An engineered derivative of sACE2 2 .v2.4-IgG1 and its glycosylation (A) Left, the structure (PDB: 6M17 ) of dimeric ACE2 (chains “A” and “B” in dark and light green) bound to RBD (gray ribbons). Glycans are shown as orange sticks. PD, protease domain; CLD, collectrin-like dimerization domain. Center and right, residues mutated to fill cavities (blue spheres), introduce disulfides (yellow spheres), or add N-glycosylation motifs (purple spheres) are shown on a single ACE2 subunit. Lead candidate sACE2 2 .S19-IgG1 has mutations V491I, M662T, N720S. (B) N-glycan types on sACE2 2 .S19-IgG1 produced in Expi293F cells. (C) Abundance of sialylated and fucosylated N-glycan structures on sACE2 2 .S19-IgG1 produced in Expi293F cells. (D) O-Glycan structures on sACE2 2 .S19-IgG1 produced in Expi293F cells following O-glycan release and MALDI-TOF-MS analysis. (E) Occupancy of the N-glycosylation sites based on glycopeptidomics analysis of sACE2 2 .S19-IgG1 from Expi293F (green), sACE2 2 .v2.4-IgG1 from Expi293F (pale gray), and sACE2 2 .v2.4-IgG1 from ExpiCHO-S (dark gray). sACE2 2 .S19-IgG1 has added glycosylation sites at positions 660 and 718. (F) Percent of the glycoforms at each N-glycosylation site that have at least one sialic acid.

Article Snippet: For measuring kinetics of sACE2 2 -IgG1/RBD interactions, responses were recorded on an Octet RED96a and analyzed with a 1:1 binding model (global fit) using instrument software (Sartorius). sACE2 2 -IgG1 proteins were immobilized at 100 nM for 60 s to AHC (anti-human IgG Fc) biosensors (Sartorius) in assay buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20, 0.5% non-fat dry milk).

Techniques: Introduce, Produced

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: BLI kinetics for monovalent binding of decoy receptors to Spike RBD

Article Snippet: For measuring kinetics of sACE2 2 -IgG1/RBD interactions, responses were recorded on an Octet RED96a and analyzed with a 1:1 binding model (global fit) using instrument software (Sartorius). sACE2 2 -IgG1 proteins were immobilized at 100 nM for 60 s to AHC (anti-human IgG Fc) biosensors (Sartorius) in assay buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20, 0.5% non-fat dry milk).

Techniques: Binding Assay, Mutagenesis

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: High sialylation and YTE mutations in the Fc region enhance the pharmacokinetics of sACE2 2 .S19-IgG1 (A) Intravenous administration of proteins at 10 mg/kg into human FcRn mice. Blood was collected into heparin via retroorbital route at the plotted time points. Plasma levels of the indicated proteins were measured by ELISA. (B and C) Proteins were injected s.c. in the flank of human FcRn mice at a dose of 10 mg/kg (solid lines) or 100 mg/kg (broken line). (B) ACE2 catalytic activity in plasma and (C) protein concentrations based on ELISA. (D) sACE2 2 .S19-IgG1(YTE) was treated with PNGase F or neuraminidase. Proteins (20 μg) were analyzed without further purification by SDS-PAGE under non-reducing conditions and stained with Coomassie. The calculated molecular weight (MW) of the mature polypeptide (excluding glycans) is 218 kD for the dimer. PNGase F has an MW of 36 kD. A. ureafaciens neuraminidase is a mixture of isoenzymes. (E) Proteins (10 μg) were analyzed by IEF gel electrophoresis. Glycosidase-treated proteins were analyzed without (−) and with (+) purification by gel filtration following treatment. (F) Purified proteins were injected i.v. at 10 mg/kg into human FcRN mice and concentrations in plasma were measured by ELISA for 5 days. For PK studies in this figure, data are mean ± SEM for n = 3 per group and proteins were purified from transiently transfected Expi293F.

Article Snippet: For measuring kinetics of sACE2 2 -IgG1/RBD interactions, responses were recorded on an Octet RED96a and analyzed with a 1:1 binding model (global fit) using instrument software (Sartorius). sACE2 2 -IgG1 proteins were immobilized at 100 nM for 60 s to AHC (anti-human IgG Fc) biosensors (Sartorius) in assay buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20, 0.5% non-fat dry milk).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Activity Assay, Purification, SDS Page, Staining, Molecular Weight, Nucleic Acid Electrophoresis, Filtration, Transfection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Pharmacokinetic properties of optimized decoy receptors in Tg32 mice

Article Snippet: For measuring kinetics of sACE2 2 -IgG1/RBD interactions, responses were recorded on an Octet RED96a and analyzed with a 1:1 binding model (global fit) using instrument software (Sartorius). sACE2 2 -IgG1 proteins were immobilized at 100 nM for 60 s to AHC (anti-human IgG Fc) biosensors (Sartorius) in assay buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20, 0.5% non-fat dry milk).

Techniques: Produced, Transfection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: A single dose of sACE2 2 .S19-IgG1(YTE) sourced from Expi293F culture protects K18-hACE2 mice from lethal SARS-CoV-2 challenge (A and B) K18-hACE2 mice were inoculated intranasally with 1 × 10 4 PFU 2019n-CoV/USA_WA1/2020 virus. Mice were administered a single i.v. dose (10 mg/kg) of sACE2 2 .S19-IgG1(YTE) (red; purified from transiently transfected Expi293F culture) or IgG1 control (gray) 24 h post-inoculation. Survival (A) and weights (B) for N = 10 mice per treatment group. p value determined by Gehan-Breslow-Wilcoxon test. (C and D) Lungs of inoculated mice were harvested at day 7 and relative viral loads were determined by qPCR for mRNA expression levels of SARS-CoV-2 Spike (C) and Nsp (D). Mean ± SEM, N = 4 per treatment group. p values determined by unpaired t test.

Article Snippet: For measuring kinetics of sACE2 2 -IgG1/RBD interactions, responses were recorded on an Octet RED96a and analyzed with a 1:1 binding model (global fit) using instrument software (Sartorius). sACE2 2 -IgG1 proteins were immobilized at 100 nM for 60 s to AHC (anti-human IgG Fc) biosensors (Sartorius) in assay buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20, 0.5% non-fat dry milk).

Techniques: Virus, Purification, Transfection, Control, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Sialylation of decoy receptors purified from different sources

Article Snippet: For measuring kinetics of sACE2 2 -IgG1/RBD interactions, responses were recorded on an Octet RED96a and analyzed with a 1:1 binding model (global fit) using instrument software (Sartorius). sACE2 2 -IgG1 proteins were immobilized at 100 nM for 60 s to AHC (anti-human IgG Fc) biosensors (Sartorius) in assay buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20, 0.5% non-fat dry milk).

Techniques: Purification

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: The v2.4 and S19 mutations in ACE2 are predicted to have low immunogenicity (A) Computational analysis of calculated affinity of peptides to HLA-II allotypes. The wild-type sACE2 2 -IgG1 sequence (top row) is scanned for peptides predicted to be displayed on a common set of 14 HLA-II allotypes. In this analysis, the minimum threshold for a peptide to be considered an antigen is predicted affinity for four HLA-II allotypes (Nhits ≥4) from a set of 14 common alleles. For sACE2 2 -IgG1 derivatives, the sequence is grayed out except for the regions where mutations are introduced to highlight whether a mutation is within a predicted epitope and/or changes the probability of presentation. Peptides that were analyzed experimentally by yeast display are indicated below with dark red bars. (B) Peptides were displayed on yeast and binding to HLA-II following an HLA-DM-dependent peptide loading reaction was measured by flow cytometry. The correlation plot shows the agreement between two independent replicates measuring mean fluorescence units (MFU) for bound HLA-II. Polyserine negative control reactions are blue, positive control peptide/HLA-II pairs are orange, and ACE2 peptides are gray. (C) Yeast display measurements of HLA-II binding to ACE2 peptides is plotted from no signal (dark blue) to high binding signal (orange).

Article Snippet: For measuring kinetics of sACE2 2 -IgG1/RBD interactions, responses were recorded on an Octet RED96a and analyzed with a 1:1 binding model (global fit) using instrument software (Sartorius). sACE2 2 -IgG1 proteins were immobilized at 100 nM for 60 s to AHC (anti-human IgG Fc) biosensors (Sartorius) in assay buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20, 0.5% non-fat dry milk).

Techniques: Sequencing, Mutagenesis, Binding Assay, Flow Cytometry, Fluorescence, Negative Control, Positive Control

Journal: EMBO Molecular Medicine

Article Title: An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS‐CoV ‐2

doi: 10.15252/emmm.202216109

Figure Lengend Snippet: A K18‐hACE2 transgenic mice were inoculated with SARS‐CoV‐2 isolate /Japan/TY7‐503/2021 (gamma variant) at 1 × 10 4 PFU. sACE2 2 .v2.4‐IgG1 (7.5 ml at 8.3 mg/ml in PBS) was delivered to the mice by a nebulizer in 25 min at 12 h, 48 h, and 84 h postinoculation. PBS was aerosol delivered as control. B, C Survival (B) and weight loss (C). N = 10 mice for each group. The P‐value of the survival curve by the Gehan–Breslow–Wilcoxon test is shown. Error bars for mouse weight are centered on the mean and show SEM. D Viral load in the lung was measured by RT–qPCR on Day 7. The mRNA expression levels of SARS‐CoV‐2 Spike, Nsp, and Rdrp are normalized to the housekeeping gene peptidylprolyl isomerase A (Ppia). Data are presented as mean ± SEM, N = 4 mice per group. * P < 0.05 by the unpaired Student's t ‐test with two‐sided. E Cytokine expression levels of Tnfa, Ifng, Il1a, and Il1b were measured by RT–qPCR normalized by Ppia. Data are presented as mean ± SEM, N = 4 mice per group. * P < 0.05 by the unpaired Student's t ‐test with two‐sided. F, G Representative H&E staining of lung sections on Day 7 postinoculation for control PBS group (F) and inhalation of the sACE2 2 .v2.4‐IgG1 group (G). Images at left are low magnifications. Boxed regions (black) are shown at higher magnification on the right. Lungs from 4 independent mice were sectioned, stained, and imaged.

Article Snippet: Plasmids for sACE2 2 ‐IgG1 (Addgene #154104), sACE2 2 .v2.4‐IgG1 (#154106), sACE2(18‐615)‐8his (#149268), sACE2(18‐615).v2.4‐8his (#149664), and Wuhan RBD‐8his (#145145) are available from Addgene.

Techniques: Transgenic Assay, Variant Assay, Aerosol, Control, Quantitative RT-PCR, Expressing, Staining

Journal: EMBO Molecular Medicine

Article Title: An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS‐CoV ‐2

doi: 10.15252/emmm.202216109

Figure Lengend Snippet: Cytokine expression (transcript levels relative to Ppia).

Article Snippet: Plasmids for sACE2 2 ‐IgG1 (Addgene #154104), sACE2 2 .v2.4‐IgG1 (#154106), sACE2(18‐615)‐8his (#149268), sACE2(18‐615).v2.4‐8his (#149664), and Wuhan RBD‐8his (#145145) are available from Addgene.

Techniques: Expressing

Journal: EMBO Molecular Medicine

Article Title: An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS‐CoV ‐2

doi: 10.15252/emmm.202216109

Figure Lengend Snippet: A A 7‐Methoxycoumarin‐4‐acetyl (MCA) conjugated peptide is quenched by a 2,4‐dinitrophenyl group. ACE2‐catalyzed cleavage of the peptide is measured by increased MCA fluorescence. Mutations H374N and H378N generate a catalytically dead sACE2 2 .v2.4‐IgG1 protein. B, C Catalytically active sACE2 2 .v2.4‐IgG1 (B) and catalytically dead sACE2 2 .v2.4(NN)‐IgG1 (C) were immobilized on BLI biosensors that were transferred to solutions of gamma RBD as the soluble analyte (0–60 s) and returned to buffer to measure dissociation (60–360 s). RBD concentrations are indicated on the right of the sensorgrams. D, E Catalytically active sACE2 2 .v2.4‐IgG1 and catalytically dead sACE2 2 .v2.4(NN)‐IgG1 were aerosolized (7.5 ml protein at 8.3 mg/ml in 25 min) and delivered by inhalation to K18‐hACE2 transgenic mice at 12 h, 48 h, and 84 h postinoculation with SARS‐CoV‐2 gamma variant. 10 mice in each group were observed for survival (D) and weight loss (E). The P ‐value of the survival curve by the Gehan–Breslow–Wilcoxon test is shown. Error bars for mouse weight are centered on the mean and show SEM. Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Fig . F Viral load in the lung was measured by RT–qPCR on Day 7. The mRNA expression levels of SARS‐CoV‐2 Spike, Nsp, and Rdrp are normalized to Ppia. Data are presented as mean ± SEM, N = 4 mice per group. ns, no significance by the unpaired Student's t ‐test with two‐sided.

Article Snippet: Plasmids for sACE2 2 ‐IgG1 (Addgene #154104), sACE2 2 .v2.4‐IgG1 (#154106), sACE2(18‐615)‐8his (#149268), sACE2(18‐615).v2.4‐8his (#149664), and Wuhan RBD‐8his (#145145) are available from Addgene.

Techniques: Fluorescence, Transgenic Assay, Variant Assay, Control, Quantitative RT-PCR, Expressing

Journal: EMBO Molecular Medicine

Article Title: An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS‐CoV ‐2

doi: 10.15252/emmm.202216109

Figure Lengend Snippet: A K18‐hACE2 transgenic mice were inoculated with 1 × 10 4 PFU SARS‐CoV‐2 WA‐1/2020 (original variant). Treatment was delayed by 24 h to allow initial virus replication and evaluate the effects of ACE2 catalytic activity at later stages of the disease. sACE2 2 .v2.4‐IgG1 was intravenously administered at 15 mg/kg, once per day for 7 days. B, C Survival (B) and weight loss (C) following treatment with a IgG1 isotype control (red) versus catalytically active (blue) and dead (green) sACE2 2 .v2.4‐IgG1. N = 8 mice for the IgG1 group and catalytic active group, N = 9 for the catalytic dead group. Error bars for mouse weight are centered on the mean and show SEM. The Gehan–Breslow–Wilcoxon test was applied to analyze the survival curves. P = 0.0099 for the catalytic active group compared with the catalytic dead group. P = 0.017 for the catalytic dead group compared with IgG1 control. D, E Catalytically active sACE2 2 .v2.4‐IgG1 (D) and catalytically dead sACE2 2 .v2.4(NN)‐IgG1 (E) were immobilized on BLI biosensors and transferred to solutions of RBD (original variant) as the soluble analyte (0–60 s) and returned to buffer to measure dissociation (60–360 s). RBD concentrations are indicated on the right of the BLI sensorgrams.

Article Snippet: Plasmids for sACE2 2 ‐IgG1 (Addgene #154104), sACE2 2 .v2.4‐IgG1 (#154106), sACE2(18‐615)‐8his (#149268), sACE2(18‐615).v2.4‐8his (#149664), and Wuhan RBD‐8his (#145145) are available from Addgene.

Techniques: Transgenic Assay, Variant Assay, Virus, Activity Assay, Control

Journal: EMBO Molecular Medicine

Article Title: An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS‐CoV ‐2

doi: 10.15252/emmm.202216109

Figure Lengend Snippet: A Binding of monomeric sACE2‐8his proteins to Expi293F cells expressing S of BA.1 omicron was measured by flow cytometry. Data are mean ± SEM, N = 4 independent experiments. B Binding of dimeric sACE2 2 ‐IgG1 proteins was compared with the binding of antibodies authorized for therapeutic use in COVID‐19 patients. Binding to Expi293F cells expressing BA.1 omicron S was measured by flow cytometry. Data are mean ± SEM, N = 4 independent biological replicates. C Neutralization of BA.1 omicron pseudovirus. sACE2 2 ‐IgG1 (gray) or sACE2 2 .v2.4‐IgG1 (blue) were incubated with pseudovirus for 1 h before adding to HeLa‐hACE2‐11 cells. Infection 48 h later was measured by luciferase reporter gene expression. Data are mean ± SD, N = 3 independent biological replicates. D, E Authentic BA.1 omicron virus (isolate USA/MD‐HP20874/2021) was incubated with sACE2 2 ‐IgG1 (D) or sACE2 2 .v2.4‐IgG1 (E) for 1 h and added to Calu‐3 cells. Infection 48 h later was measured by RT–qPCR for the viral N gene. 3 μM remdesivir (black columns) is a positive neutralization control. Data are mean ± SD, N = 4 independent replicates.

Article Snippet: Plasmids for sACE2 2 ‐IgG1 (Addgene #154104), sACE2 2 .v2.4‐IgG1 (#154106), sACE2(18‐615)‐8his (#149268), sACE2(18‐615).v2.4‐8his (#149664), and Wuhan RBD‐8his (#145145) are available from Addgene.

Techniques: Binding Assay, Expressing, Flow Cytometry, Neutralization, Incubation, Infection, Luciferase, Gene Expression, Virus, Quantitative RT-PCR, Control

Journal: EMBO Molecular Medicine

Article Title: An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS‐CoV ‐2

doi: 10.15252/emmm.202216109

Figure Lengend Snippet: A K18‐hACE2 transgenic mice were inoculated intranasally with 1 × 10 6 PFU SARS‐CoV‐2 BA.1 omicron (isolate USA/MD‐HP20874/2021) and treated 24 h postinoculation with IgG1 isotype control or sACE2 2 .v2.4‐IgG1 (15 mg/kg/day intravenously, once per day for 7 days). B, C Survival (B) and weight loss (C). N = 8 (IgG1 control) or 9 (sACE2 2 .v2.4‐IgG1 treated) mice. Error bars for mouse weight are centered on the mean and show SEM. D Maximal weight loss of mice (compared with Day 0) within 14 days following BA.1 omicron virus infection. Data are presented as mean ± SEM, N = 8 (IgG1 control) or 9 (sACE2 2 .v2.4‐IgG1 treated) mice. ** P < 0.01 by two‐ways ANOVA, corrected by Tukey. E Viral Spike, Nsp, and Rdrp mRNA levels in lung tissue were measured by RT–qPCR on Days 8 and 14, normalized to Ppia transcript levels. Data are presented as mean ± SEM, N = 4 mice per group. ns, not significant; ** P < 0.01 by two‐ways ANOVA, corrected by Tukey.

Article Snippet: Plasmids for sACE2 2 ‐IgG1 (Addgene #154104), sACE2 2 .v2.4‐IgG1 (#154106), sACE2(18‐615)‐8his (#149268), sACE2(18‐615).v2.4‐8his (#149664), and Wuhan RBD‐8his (#145145) are available from Addgene.

Techniques: Transgenic Assay, Control, Virus, Infection, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS‐CoV ‐2

doi: 10.15252/emmm.202216109

Figure Lengend Snippet: A Using flow cytometry, binding to Expi293F cells expressing S of BA.2 omicron was measured for monomeric sACE2‐8his proteins. Data are mean ± SEM, N = 3 independent biological replicates. B Binding of antibodies and dimeric sACE2 2 ‐IgG1 proteins to Expi293F cells expressing BA.2 omicron S measured by flow cytometry. Data are mean ± SEM, N = 4 independent experiments. C BA.2 omicron pseudovirus was incubated with sACE2 2 ‐IgG1 (gray) or sACE2 2 .v2.4‐IgG1 (blue) for 1 h and added to HeLa‐hACE2‐11 cells. Infection was measured after 48 h. Data are mean ± SD, N = 3 independent replicates. D, E Binding of monomeric sACE2‐8his proteins (D) and dimeric IgG1 proteins (E) to Expi293F cells expressing BA.4/BA.5 omicron S as measured by flow cytometry. Data are mean ± SEM, N = 3 independent experiments.

Article Snippet: Plasmids for sACE2 2 ‐IgG1 (Addgene #154104), sACE2 2 .v2.4‐IgG1 (#154106), sACE2(18‐615)‐8his (#149268), sACE2(18‐615).v2.4‐8his (#149664), and Wuhan RBD‐8his (#145145) are available from Addgene.

Techniques: Flow Cytometry, Binding Assay, Expressing, Incubation, Infection

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Elucidating Design Principles for Engineering Cell‐Derived Vesicles to Inhibit SARS‐CoV‐2 Infection

doi: 10.1002/smll.202200125

Figure Lengend Snippet: Decoy vesicles are robust to drug‐resistant Spike mutations. A) Cartoon depicting the SARS‐CoV‐2 Spike variants investigated in this study and their relative susceptibility to inhibition by soluble ACE2 or monoclonal antibody treatments. B) Top: Dose‐response curves depicting the inhibition of various strains of SARS‐CoV‐2 Spike pseudotyped lentivirus against soluble ACE2, WT‐ACE2 UC‐EVs, and Mut‐ACE2 UC‐EVs. Curves are normalized to the percent of cells transduced at the lowest EV dose in a particular curve. Symbols represent the mean of three biological replicates; error bars are standard error of the mean. Data are representative of two independent experiments. B) Bottom: Log(ID 50 ) values calculated from the data in the top portion of this panel. Numbers above each point report the relative resistance for a given strain relative to the parental (D614G) strain for that particular inhibitor treatment. Error bars represent 95% confidence intervals for the parameter (ID 50 ) estimation.

Article Snippet: SACE2 (Sino Biological, 10108‐H08H) was resuspended in sterile, nuclease‐free H 2 O at a stock concentration of 0.25 mg mL −1 per manufacturer instructions and then diluted further in DMEM prior to inhibition experiments.

Techniques: Inhibition