Emily McNally, Christian Yee, Mike Maloney, Devin Bridgen, Jonathan Gilbert, Marija Tadin-Strapps, Armon Sharei, and Jacquelyn Hanson
Publication text
Advantages of the Cell Squeeze® Technology for Multiplex Gene Editing Emily McNally, Christian Yee, Mike Maloney, Devin Bridgen, Jonathan Gilbert, Marija Tadin-Strapps, Armon Sharei, and Jacquelyn Hanson SQZ Biotechnologies, Watertown MA
Introduction
Cell Squeeze® Technology Enables Efficient Primary Immune Cell Editing with Preserved Function
- Cell Squeeze® technology can efficiently deliver a variety of cargo types including proteins,
peptides, nucleic acids, ribonucleoprotein (RNP) complexes, and large molecules including whole AAVs.
- Co-delivery of multiple cargoes of the same or different molecule type may be delivered in a single
mechanical deformation with minimal loss in delivery efficiency of either cargo.
- We developed a sequential squeezing method in which cells may be repeatedly squeezed in order
to introduce multiple cargoes with increased efficiency, preservation of cell function, and desired phenotype.
- By using mechanical deformation via Cell Squeeze® technology, we can achieve efficient multigene editing with minimal perturbation of gene expression or cytokine secretion, and preserved T
cell function in vitro. Complex Cell Engineering Capabilities Enabled by the Cell Squeeze® Technology
- With our microfluidic approach to intracellular delivery, cell membranes are deformed as the
cells pass through constrictions in channels, resulting in payload diffusion from the surrounding buffer directly into the cytosol (1).
- The translational potential and scalability of cell-based therapies are often limited by
complications related to effectively engineering and manufacturing functional cells, including low efficiency, cytotoxicity, and off-target effects (2).
- Cell Squeeze® technology reduces cell toxicity and off-target effects observed with conventional
delivery methods by eliminating the need for electrical fields or exogenous vectors such as viruses, differentiating our approach as potentially transformative for enabling cell therapies that have been historically challenging to engineer. Cell Squeeze® Technology Squeeze Process: Cells flow through parallel microfluidic channels and are forced through a constriction point smaller than the cell diameter. This process results in temporary disruption of the cell membrane to facilitate delivery. Scalable for High Throughput: We can treat more than 1010 cells/minute. Cell Squeeze® Technology Enables Efficient Multiplex Genome Engineering Through Sequential EditingSqueeze RNP only mRNA only RNP + mRNA % of T cells
B2m+cd86-
B2m+cd86+
B2m-cd86+
B2m-cd86-
Multiplexed B2M RNP + CD86 mRNA RNP only mRNA only RNP + mRNA
B2m
Cd86
High Efficiency Multiplex Engineering of Cells and Materials Using Cell Squeeze® Technology0 TIM3 Surface Expression, Day 7
% Tim3+
*0 CD3 (TRAC proxy) Surface Expression, Day 7
% Cd3+
No Contact Squeeze Control
B2m Rnp
Tim-3 Rnp
Trac Rnp
Multiplex RNP Sequential RNP *, p<0.05 **, p<0.01 ***, p<0.001 ****, p<0.0001 compared to squeeze controlUntreated SQZ Control Multiplex RNP Sequential RNP Multiple Edits per Cell % of T cells No-Negative Single-Negative Double-Negative Triple-Negative
Summary
- Sharei A, et al. PNAS, 2013 Feb 5;110(6):2082-7
- Zhang M, et al. J Immunol Methods, 2014 Jun;408:123-131.
Contact Information Jacquelyn Hanson, PhD, SQZ Biotechnologies, jacquelyn.hanson@sqzbiotech.com References0 1 2 3 4 Sequential Live Cell Counts Day Cells (x106) Squeeze control
Rnp Sqz
Squeeze 1 Squeeze 2 Squeeze 3 Unstimulated Human T cells
B2m Rnp +
CD86 mRNA
Cd3/cd28
Activated Human T cells Day 0 Day 2 Day 3
Trac Rnp B2m Rnp Tim-3 Rnp
Day 0 B2M, TIM-3, TRAC RNPs Cell Squeeze® technology enables delivery of genome engineering material to primary T cells with minimal effect on cell phenotype and health
- Squeezed cells perform more similarly to untreated cells in vitro in terms of gene expression profiles,
cytokine production, and expansion rate.
- In contrast, electroporated cells exhibit large changes to gene expression which include cytokine
upregulation and delays in expansion.
- Similar Squeeze performance and characteristics in a variety of cell types suggests broad applicability.
Multiplexed editing is efficiently achieved through sequential squeezes
- Both multiplexed and sequential applications of Cell Squeeze® technology allow for broad uses in
genome engineering.
- Sequential application of genome editing reduces potential for risk of multiple simultaneous cuts
with minimal effect on cell heath and expansion. Human T Cell Proliferation is Unaffected by Cell Squeeze® Technology but Delayed Following Electroporation0 1×105 1×106 1×107 1×108 1×109 1×1010 Days Post-delivery Normalized cell count ** * Electroporation v Squeeze *, p<0.05 **, p<0.01 ***, p<0.001 ****, p<0.0001
*il-10
Gm-csf
IFNy IL-12(p70)
Il-2
Il-5
Il-13
Il-17a
Il-4
Il-6
Il-8
TNFa Condition 1 Condition 2 Condition 1 Condition 2 UnstimulatedActivated pg/mL 50000 B2M Surface Expression, Day 7
% B2m+
Unstimulated human T cells Cell Squeeze® Technology Electroporation MAPRE3
Tspan
Emp1
Lmna1
Cd109
Cd200
Ifng
Cxcl9
Il2
Cell Squeeze® Technology Preserves Cell Phenotype in Contrast to Dramatic Effects of Electroporation5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8 -0.9 -1.0 -1.1 -1.2 -1.3 -1.4 -1.5 -1.6 -1.7 -1.8 -1.9 -2.0 -2.1 -2.2 -2.3 -2.4 -2.5 -2.6 -2.7 -2.8 -2.9 -3.0 -3.1 -3.2 -3.3 -3.4 -3.5 -3.6 -3.7 -3.8 -3.9 -4.0 -4.1 -4.2 -4.3 -4.4 -4.5 -4.6 -4.7 -4.8 -4.9-5.0 log2Fold change -5 EP Squeeze *, p <0.05; **, p <0.01, compared to respective squeeze Cytokines/chemokines ElectroporationSqueezesqueeze electroporation Fold change (rel to control)Untreated Electoproation + RNP Squeeze + RNP 150 IFNy Relative fold change (ΔΔCt) **Untreated Electoproation + RNP Squeeze + RNP
500 Il-2
Relative fold change (ΔΔCt)squeeze
Ifn
*squeeze
Il-9
*squeeze
Il-2
Cell Squeeze® Technology Preserves Cell Function While Electroporation Non-specifically Activates T Cells Gene Expression Changes are Greater Following Electroporation than Squeeze Gene Misregulation Persists Following Electroporation No cargo Gene Expression Array qPCR analysis Electroporation Results in Broad Pathway Upregulation While Squeeze Induces Only the Pathway Involved in Membrane Repair Multiplex ELISA *, p <0.05; to squeeze No Disruption in Cytokine Secretion Following Squeeze Unstimulated human T cells squeezed and maintained unstimulated or activated on CD3/CD28 beads Activated Human T Cells Proliferate Similarly to Untreated Sequencing Validates High Level of Multiplex Editing for Sequentially Squeezed RNPs No Adverse Effect on Editing Efficiency in Multi-Edited Compared to Single RNP Surface Knock Down Achieved More Efficiently Through Sequential Squeezing with No Adverse Effect on Proliferation Counts taken prior to squeeze 10X Genomics analysis Predicted (baseline) reads per cell Predicted (baseline) reads per cell Actual reads per cell Actual reads per cell Sequential RNP Squeeze Multiplex RNP Squeeze Multi-Editing *, p <0.01; **, p <0.001; ***, p <1e-6
Egfp
T cells (CD3+) B cells (CD19+) Monocytes (CD14+) NK cells (CD56+) Empty EGFP mRNA mRNA Delivery to all Major Subsets of Human PBMCs in a Single Treatment Simultaneous Gene Editing and mRNA Expression Achieved in T Cells Broad Applicability of Genome Engineering Beyond T Cells 6 hours Electroporation 24 hours Gene Misregulation Also Observed Following EP in CD34+ Cells High Editing Efficiency Achieved in CD34+ Colony Formation Assay Day 0 Day 1 Thaw human donor CD34+ cells … Week 2 End of CFC Assay Deliver RNP and immediately plate cells Control Squeeze
+ Rnp5.0
4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8 -0.9 -1.0 -1.1 -1.2 -1.3 -1.4 -1.5 -1.6 -1.7 -1.8 -1.9 -2.0 -2.1 -2.2 -2.3 -2.4 -2.5 -2.6 -2.7 -2.8 -2.9 -3.0 -3.1 -3.2 -3.3 -3.4 -3.5 -3.6 -3.7 -3.8 -3.9 -4.0 -4.1 -4.2 -4.3 -4.4 -4.5 -4.6 -4.7 -4.8 -4.9-5.0 log2Fold change -5
Asgct
May 11-14, 2021
Some publications reference SQZ Biotechnologies, a company Dr. Armon Sharei and colleagues previously founded. Portal is the next generation company leveraging Dr. Sharei's platform technology.
# Advantages of the Cell Squeeze® Technology for Multiplex Gene Editing
This page is a text version of a Portal Biotechnologies publication. The bibliographic record is labeled in the text so it can be read without the surrounding website.
## Bibliographic record
- Title: Advantages of the Cell Squeeze® Technology for Multiplex Gene Editing
- Document type: Poster
- Year: 2021
- Outlet: ASGCT
- Page URL: https://portal.bio/publications/2021-advantages-of-the-cell-squeeze-technology-for-multiplex-gene-editing--ajkG4xEAACwAc9_i
- Markdown URL: https://portal.bio/publications/2021-advantages-of-the-cell-squeeze-technology-for-multiplex-gene-editing--ajkG4xEAACwAc9_i.md
- Original file: https://portal-bio.cdn.prismic.io/portal-bio/65cce83c9be9a5b998b5d20a_ASGCT-2021-Platform-Hanson-FINAL-updatedJul2022.pdf
## Summary
Emily McNally, Christian Yee, Mike Maloney, Devin Bridgen, Jonathan Gilbert, Marija Tadin-Strapps, Armon Sharei, and Jacquelyn Hanson
## Publication text
Advantages of the Cell Squeeze® Technology for Multiplex Gene Editing Emily McNally, Christian Yee, Mike Maloney, Devin Bridgen, Jonathan Gilbert, Marija Tadin-Strapps, Armon Sharei, and Jacquelyn Hanson SQZ Biotechnologies, Watertown MA
### Introduction
Cell Squeeze® Technology Enables Efficient Primary Immune Cell Editing with Preserved Function
- Cell Squeeze® technology can efficiently deliver a variety of cargo types including proteins,
peptides, nucleic acids, ribonucleoprotein (RNP) complexes, and large molecules including whole AAVs.
- Co-delivery of multiple cargoes of the same or different molecule type may be delivered in a single
mechanical deformation with minimal loss in delivery efficiency of either cargo.
- We developed a sequential squeezing method in which cells may be repeatedly squeezed in order
to introduce multiple cargoes with increased efficiency, preservation of cell function, and desired phenotype.
- By using mechanical deformation via Cell Squeeze® technology, we can achieve efficient multigene editing with minimal perturbation of gene expression or cytokine secretion, and preserved T
cell function in vitro. Complex Cell Engineering Capabilities Enabled by the Cell Squeeze® Technology
- With our microfluidic approach to intracellular delivery, cell membranes are deformed as the
cells pass through constrictions in channels, resulting in payload diffusion from the surrounding buffer directly into the cytosol (1).
- The translational potential and scalability of cell-based therapies are often limited by
complications related to effectively engineering and manufacturing functional cells, including low efficiency, cytotoxicity, and off-target effects (2).
- Cell Squeeze® technology reduces cell toxicity and off-target effects observed with conventional
delivery methods by eliminating the need for electrical fields or exogenous vectors such as viruses, differentiating our approach as potentially transformative for enabling cell therapies that have been historically challenging to engineer. Cell Squeeze® Technology Squeeze Process: Cells flow through parallel microfluidic channels and are forced through a constriction point smaller than the cell diameter. This process results in temporary disruption of the cell membrane to facilitate delivery. Scalable for High Throughput: We can treat more than 1010 cells/minute. Cell Squeeze® Technology Enables Efficient Multiplex Genome Engineering Through Sequential EditingSqueeze RNP only mRNA only RNP + mRNA % of T cells
### B2m+cd86-
### B2m+cd86+
### B2m-cd86+
### B2m-cd86-
Multiplexed B2M RNP + CD86 mRNA RNP only mRNA only RNP + mRNA
### B2m
### Cd86
High Efficiency Multiplex Engineering of Cells and Materials Using Cell Squeeze® Technology0 TIM3 Surface Expression, Day 7
### % Tim3+
*0 CD3 (TRAC proxy) Surface Expression, Day 7
### % Cd3+
No Contact Squeeze Control
### B2m Rnp
### Tim-3 Rnp
### Trac Rnp
Multiplex RNP Sequential RNP *, p<0.05 **, p<0.01 ***, p<0.001 ****, p<0.0001 compared to squeeze controlUntreated SQZ Control Multiplex RNP Sequential RNP Multiple Edits per Cell % of T cells No-Negative Single-Negative Double-Negative Triple-Negative
### Summary
1. Sharei A, et al. PNAS, 2013 Feb 5;110(6):2082-7
2. Zhang M, et al. J Immunol Methods, 2014 Jun;408:123-131.
Contact Information Jacquelyn Hanson, PhD, SQZ Biotechnologies, jacquelyn.hanson@sqzbiotech.com References0 1 2 3 4 Sequential Live Cell Counts Day Cells (x106) Squeeze control
### Rnp Sqz
Squeeze 1 Squeeze 2 Squeeze 3 Unstimulated Human T cells
### B2m Rnp +
CD86 mRNA
### Cd3/cd28
Activated Human T cells Day 0 Day 2 Day 3
### Trac Rnp B2m Rnp Tim-3 Rnp
Day 0 B2M, TIM-3, TRAC RNPs Cell Squeeze® technology enables delivery of genome engineering material to primary T cells with minimal effect on cell phenotype and health
- Squeezed cells perform more similarly to untreated cells in vitro in terms of gene expression profiles,
cytokine production, and expansion rate.
- In contrast, electroporated cells exhibit large changes to gene expression which include cytokine
upregulation and delays in expansion.
- Similar Squeeze performance and characteristics in a variety of cell types suggests broad applicability.
Multiplexed editing is efficiently achieved through sequential squeezes
- Both multiplexed and sequential applications of Cell Squeeze® technology allow for broad uses in
genome engineering.
- Sequential application of genome editing reduces potential for risk of multiple simultaneous cuts
with minimal effect on cell heath and expansion. Human T Cell Proliferation is Unaffected by Cell Squeeze® Technology but Delayed Following Electroporation0 1×105 1×106 1×107 1×108 1×109 1×1010 Days Post-delivery Normalized cell count ** * Electroporation v Squeeze *, p<0.05 **, p<0.01 ***, p<0.001 ****, p<0.0001
### *il-10
### Gm-csf
IFNy IL-12(p70)
### Il-2
### Il-5
### Il-13
### Il-17a
### Il-4
### Il-6
### Il-8
TNFa Condition 1 Condition 2 Condition 1 Condition 2 UnstimulatedActivated pg/mL 50000 B2M Surface Expression, Day 7
### % B2m+
Unstimulated human T cells Cell Squeeze® Technology Electroporation MAPRE3
### Tspan
### Emp1
### Lmna1
### Cd109
### Cd200
### Ifng
### Cxcl9
### Il2
Cell Squeeze® Technology Preserves Cell Phenotype in Contrast to Dramatic Effects of Electroporation5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8 -0.9 -1.0 -1.1 -1.2 -1.3 -1.4 -1.5 -1.6 -1.7 -1.8 -1.9 -2.0 -2.1 -2.2 -2.3 -2.4 -2.5 -2.6 -2.7 -2.8 -2.9 -3.0 -3.1 -3.2 -3.3 -3.4 -3.5 -3.6 -3.7 -3.8 -3.9 -4.0 -4.1 -4.2 -4.3 -4.4 -4.5 -4.6 -4.7 -4.8 -4.9-5.0 log2Fold change -5 EP Squeeze *, p <0.05; **, p <0.01, compared to respective squeeze Cytokines/chemokines ElectroporationSqueezesqueeze electroporation Fold change (rel to control)Untreated Electoproation + RNP Squeeze + RNP 150 IFNy Relative fold change (ΔΔCt) **Untreated Electoproation + RNP Squeeze + RNP
### 500 Il-2
Relative fold change (ΔΔCt)squeeze
### Ifn
*squeeze
### Il-9
*squeeze
### Il-2
Cell Squeeze® Technology Preserves Cell Function While Electroporation Non-specifically Activates T Cells Gene Expression Changes are Greater Following Electroporation than Squeeze Gene Misregulation Persists Following Electroporation No cargo Gene Expression Array qPCR analysis Electroporation Results in Broad Pathway Upregulation While Squeeze Induces Only the Pathway Involved in Membrane Repair Multiplex ELISA *, p <0.05; to squeeze No Disruption in Cytokine Secretion Following Squeeze Unstimulated human T cells squeezed and maintained unstimulated or activated on CD3/CD28 beads Activated Human T Cells Proliferate Similarly to Untreated Sequencing Validates High Level of Multiplex Editing for Sequentially Squeezed RNPs No Adverse Effect on Editing Efficiency in Multi-Edited Compared to Single RNP Surface Knock Down Achieved More Efficiently Through Sequential Squeezing with No Adverse Effect on Proliferation Counts taken prior to squeeze 10X Genomics analysis Predicted (baseline) reads per cell Predicted (baseline) reads per cell Actual reads per cell Actual reads per cell Sequential RNP Squeeze Multiplex RNP Squeeze Multi-Editing *, p <0.01; **, p <0.001; ***, p <1e-6
### Egfp
T cells (CD3+) B cells (CD19+) Monocytes (CD14+) NK cells (CD56+) Empty EGFP mRNA mRNA Delivery to all Major Subsets of Human PBMCs in a Single Treatment Simultaneous Gene Editing and mRNA Expression Achieved in T Cells Broad Applicability of Genome Engineering Beyond T Cells 6 hours Electroporation 24 hours Gene Misregulation Also Observed Following EP in CD34+ Cells High Editing Efficiency Achieved in CD34+ Colony Formation Assay Day 0 Day 1 Thaw human donor CD34+ cells … Week 2 End of CFC Assay Deliver RNP and immediately plate cells Control Squeeze
### + Rnp5.0
4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8 -0.9 -1.0 -1.1 -1.2 -1.3 -1.4 -1.5 -1.6 -1.7 -1.8 -1.9 -2.0 -2.1 -2.2 -2.3 -2.4 -2.5 -2.6 -2.7 -2.8 -2.9 -3.0 -3.1 -3.2 -3.3 -3.4 -3.5 -3.6 -3.7 -3.8 -3.9 -4.0 -4.1 -4.2 -4.3 -4.4 -4.5 -4.6 -4.7 -4.8 -4.9-5.0 log2Fold change -5
### Asgct
May 11-14, 2021
## Provenance
Some publications reference SQZ Biotechnologies, a company Dr. Armon Sharei and colleagues previously founded. Portal is the next generation company leveraging Dr. Sharei's platform technology.
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