PAIRED-CELL PROFILING SYSTEMS FOR HIGH-THROUGHPUT SCREENING SYNCYTIUM FORMATION
Provided is a high throughput screening method for identifying cell fusion or syncytium formation potential. In particular, the cell fusion or syncytium formation potential of a viral spike protein. Also provided is a method for identifying a cellular factor that promotes spike protein-induced syncytium formation. Also provided is a method of inhibiting or suppressing syncytium formation induced by a viral spike protein.
1 . A high throughput screening method for identifying cell fusion or syncytium formation potential of a viral spike protein, the method comprising the step of
(a) preparing a plurality of spike protein variants;
(b) incorporating said spike protein variants into a plurality of spike-presenting sender cells, each cell comprising a spike protein variant from said plurality of spike protein variants;
(c) contacting said spike-presenting sender cells with a plurality of receiver cells expressing angiotensin-converting enzyme 2 (ACE2) to obtain a mixture;
(d) incubating the mixture obtained in step (c) in a microfluidic system comprising multiple droplets, each droplet comprising an individual sender cell and an individual receiver cell; and
(e) determining the syncytium-forming potential of the spike protein variants in the droplets.
2 . The method of claim 1 , wherein the viral spike protein is a SARS-COV-2 (COVID-19) spike protein.
3 . The method of claim 1 , wherein the plurality of spike protein variants is prepared by site saturation mutagenesis (SSM).
4 . The method of claim 3 , wherein the SSM is over the fusion peptide proximal region (FPPR) or the furin cleavage site of SARS-COV-2.
5 . The method of claim 1 , wherein the syncytium-forming potential of the spike protein variants is determined by measuring a signal generated upon fusion of the sender and receiver cells.
6 . The method claim 5 , wherein the signal is a fluorescent, bioluminescent, chemiluminescent or a radioactive signal.
7 . The method claim 5 , wherein the signal is a green fluorescent protein (GFP) signal that is generated upon fusion of the sender and receiver cells.
8 . The method of claim 1 , wherein the syncytium-forming potential of the spike protein variants is determined using deep mutational scanning (DMS) in the spike-presenting sender cells.
9 . The method of claim 1 , further comprising the step of enriching the spike variants having enhanced syncytium-formation potential.
10 . The method of claim 1 , further comprising the step of analyzing the abundance of each variant by sequencing spike variants having enhanced syncytium-formation potential.
11 . A high throughput screening method for identifying cell fusion or syncytium formation potential of a viral spike protein, the method comprising the step of
(a) preparing a plurality of spike protein variants;
(b) incorporating said spike protein variants into a plurality of spike-presenting sender cells, each cell comprising a spike protein variant from said plurality of spike protein variants;
(c) contacting said spike-presenting sender cells with a plurality of receiver cells expressing angiotensin-converting enzyme 2 (ACE2) to obtain a mixture;
(d) performing size-exclusion selection by passing the mixture through a cell strainer; and
(e) determining the syncytium-forming potential of the spike protein variants.
12 . The method of claim 11 , further comprising the step of collecting a first syncytia population that is retained on said strainer, and a second syncytia population that passes through the strainer.
13 . The method of claim 12 , further comprising the step of enriching the spike variants having enhanced syncytium-formation potential from the first and second syncytia populations.
14 . The method of claim 12 , further comprising the step of analyzing the abundance of each variant by sequencing spike variants having enhanced syncytium-formation potential in the first and second syncytia populations.
15 . The method of claim 11 , wherein the plurality of spike protein variants is prepared by site saturation mutagenesis (SSM).
16 . The method of claim 15 , wherein the SSM is over the fusion peptide proximal region (FPPR) or the furin cleavage site of SARS-COV-2.
17 . The method of claim 11 , wherein the syncytium-forming potential of the spike protein variants is determined by measuring a signal generated upon fusion of the sender and receiver cells.
18 . The method of claim 17 , wherein the signal is a fluorescent, bioluminescent, chemiluminescent or a radioactive signal.
19 . The method of claim 17 , wherein the signal is a green fluorescent protein (GFP) signal that is generated upon fusion of the sender and receiver cells.
20 . The method of claim 11 , wherein the syncytium-forming potential of the spike protein variants is determined using deep mutational scanning (DMS) in the spike-presenting sender cells.
21 . A method for identifying a cellular factor that promotes viral spike protein-induced syncytium formation, the method comprising the steps of:
(a) preparing a plurality of spike protein variants;
(b) incorporating said spike protein variants into a plurality of spike-presenting sender cells, each cell comprising a spike protein variant from said plurality of spike protein variants;
(c) contacting said spike-presenting sender cells with a plurality of receiver cells expressing angiotensin-converting enzyme 2 (ACE2) and single guide RNA (sgRNA)-Cas9 to obtain a mixture;
(d) isolating unfused receiver cells from the mixture;
(e) comparing the sgRNA abundance in unmixed receiver cells and the isolated unfused receiver cells from step (d); and
(f) identifying a factor in the receiver cell that promotes syncytium formation.
22 . The method of claim 21 , wherein the viral spike protein is a SARS-COV-2 (COVID-19) spike protein.
23 . The method of claim 21 , wherein the plurality of spike protein variants is prepared by site saturation mutagenesis (SSM).
24 . The method of claim 23 , wherein the SSM is over the fusion peptide proximal region (FPPR) or the furin cleavage site of SARS-COV-2.
25 . The method of claim 21 , wherein the syncytium-forming potential of the spike protein variants is determined by measuring a signal generated upon fusion of the sender and receiver cells.
26 . The method of claim 25 , wherein the signal is a fluorescent, bioluminescent, chemiluminescent or a radioactive signal.
27 . The method of claim 25 , wherein the signal is a green fluorescent protein (GFP) signal that is generated upon fusion of the sender and receiver cells.
28 . The method of claim 21 , wherein the cellular factor comprises a core regulator of clathrin-mediated endocytosis (CME).
29 . The method of claim 28 , wherein the CME regulator is selected from the group consisting of FCHO2, AP2M1, CAB39, RNF2 and GBP6.
30 . A high throughput screening method for identifying cell fusion or syncytium formation potential of a first protein and a second protein in a biological system, the method comprising the steps of:
(a) preparing a plurality of first protein variants;
(b) incorporating said first protein variants into a plurality of first protein-presenting cells, each cell comprising a first protein variant from said plurality of first protein variants;
(c) contacting said first protein presenting cells with a plurality of cells expressing said second protein to obtain a mixture;
(d) incubating the mixture obtained in step (c) in a microfluidic system comprising multiple droplets, each droplet comprising an individual first protein presenting cell and an individual cell expressing said second protein; and
(e) determining the syncytium-forming potential of the first protein variants in the droplets.
31 . The method of claim 30 , wherein the biological system is a virus, tumor, maternal-fetal material exchange in the placenta, muscle contraction and bone resorption.
32 . The method of claim 31 wherein the virus is selected from the group consisting of human immunodeficiency virus, Herpesviridae, respiratory syncytial virus and Coronaviridae.
33 . The method of claim 30 , wherein the first protein is from a tumor and the second protein is from normal somatic cells or dendritic cells.
34 . The method of claim 30 , wherein the first protein or second protein is from multinucleated cells selected from the group consisting of syncytiotrophoblasts, myotybes and osteoclasts.
35 . The method of claim 30 , wherein the first protein is from B cells and the second protein is from myeloma cells that produces hydridoma.
36 . The method of claim 30 , wherein the first protein is from human embryonic stem cells and the second protein is from somatic cells.
37 . The method of claim 30 , wherein the syncytium-forming potential of the first protein variants is determined by measuring a signal generated upon fusion of the first protein presenting cells and cells expressing said second protein.
38 . The method of claim 37 , wherein the signal is a fluorescent, bioluminescent, chemiluminescent or a radioactive signal.
39 . The method of claim 38 , wherein the signal is a green fluorescent protein (GFP) signal that is generated upon fusion of the first protein presenting cells and cells expressing said second protein.
40 . The method of claim 30 , wherein the syncytium-forming potential of the first protein variants is determined using deep mutational scanning (DMS) in the first protein-presenting cells.
41 . The method of claim 30 , further comprising the step of enriching the first protein variants having enhanced syncytium-formation potential.
42 . The method of claim 30 , further comprising the step of analyzing the abundance of each variant by sequencing first protein variants having enhanced syncytium-formation potential.
43 . The method of claim 30 , wherein the mixture in step (c) further comprises single guide RNA (sgRNA)-Cas9, and further comprising the steps of (f) isolating unfused cells expressing said second protein from the mixture; (g) comparing the sgRNA abundance in unmixed cells expressing said second protein and the isolated unfused cells expressing said second protein from step (d).
44 . A high throughput screening method for identifying cell fusion or syncytium formation potential of a first protein and a second protein in a biological system, the method comprising the step of
(a) preparing a plurality of first protein variants;
(b) incorporating said first protein variants into a plurality of first protein-presenting cells, each cell comprising a first protein variant from said plurality of first protein variants;
(c) contacting said first-presenting cells with a plurality of cells expressing a second protein to obtain a mixture;
(d) performing size-exclusion selection by passing the mixture through a cell strainer; and
(e) determining the syncytium-forming potential of the first protein variants.
45 . The method of claim 44 , further comprising the step of collecting a first syncytia population that is retained on said strainer, and a second syncytia population that passes through the strainer.
46 . The method of claim 45 , further comprising the step of enriching the first protein variants having enhanced syncytium-formation potential from the first and second syncytia populations.
47 . The method of claim 45 , further comprising the step of analyzing the abundance of each variant by sequencing the first protein variants having enhanced syncytium-formation potential in the first and second syncytia populations.
48 . The method of claim 44 , wherein the plurality of the first protein variants is prepared by site saturation mutagenesis (SSM).
49 . The method of claim 44 , wherein the syncytium-forming potential of the first protein variants is determined by measuring a signal generated upon fusion of the first protein presenting cells and cells expressing said second protein.
50 . The method of claim 49 , wherein the signal is a fluorescent, bioluminescent, chemiluminescent or a radioactive signal.
51 . The method of claim 50 , wherein the signal is a green fluorescent protein (GFP) signal that is generated upon fusion of the first protein presenting cells and cells expressing said second protein.
52 . The method of claim 44 , wherein the mixture in step (c) further comprises single guide RNA (sgRNA)-Cas9, and further comprising the steps of (f) isolating unfused cells expressing said second protein from the mixture; (g) comparing the sgRNA abundance in unmixed cells expressing said second protein and the isolated unfused cells expressing said second protein.
53 . The method of claim 44 , wherein the syncytium-forming potential of the first protein variants is determined using deep mutational scanning (DMS) in the first protein-presenting cells.