Jacquelyn Hanson, Emily McNally, Kendra Foley, Patricia Cho, Joshua Bugge, Tia DiTommaso, Jonathan Gilbert, Marija Tadin-Strapps, Howard Bernstein, and Armon Sharei
- Cells
- CD34+, T cells, NK cells
- Cargos
- Polymers, Viral Particle
Publication text
no guide gB2M-3 gB2M-4 gB2M-6 % B2M positive B2M Surface Expression ** * **no guide g3 g5 g7 g8 TRAC Surface Expression % TCRa/b positive * *SQZ Microfluidic Platform Enables Delivery of Gene Editing Materials and Transduction of Difficult to Engineer Cell Types While Preserving Cell Phenotype and Function Jacquelyn Hanson, Emily McNally, Kendra Foley, Patricia Cho, Joshua Bugge, Tia DiTommaso, Jonathan Gilbert, Marija Tadin-Strapps, Howard Bernstein, and Armon Sharei SQZ Biotechnologies, Watertown, MA, USA Cell Squeeze® Enables Efficient Primary Immune Cell Editing with Preserved Function
- The translational potential of cell-based therapies is often limited by
complications related to effectively engineering and manufacturing functional cells, including low efficiency, cytotoxicity, off-target effects, and limited scalability.
- Specifically, Zhang et al found that EP treatment of primary T cells
substantially alters cell state, including increases in intracellular calcium levels, increased T cell activation markers CD154 and CD69, and overall increases in aberrant transcriptional activity. (2)
- By using mechanical deformation via Cell Squeeze®, we can achieve
efficient genome editing with minimal perturbation of gene expression or cytokine secretion, and preserved T cell function in vitro. Cell Engineering Capabilities Enabled by the Cell Squeeze® Platform
- Our microfluidic approach to delivery, Cell Squeeze® (SQZ), mechanically
deforms cell membranes through constricting channels, resulting in payload diffusion from the surrounding buffer directly into the cytosol. (1)
- SQZ reduces cell toxicity and off-target effects observed with conventional
delivery methods by eliminating the need for electrical fields or exogenous materials such as viral vectors. These advantages differentiate SQZ as a potentially transformative platform for enabling cell therapies that have been historically challenging to engineer, including the use of CRISPR to engineer cell function.
- Cells subjected to SQZ have minimal aberrant transcriptional responses
compared to electroporation (EP) and exhibit editing efficiencies comparable to conventional methods such as EP.
- SQZ-mediated engineering of immune cells results in the desired
phenotype with minimal effect on viability, baseline gene expression, and cell function compared to EP. SQZ 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. Minimal Side Effects With SQZ Platform in Contrast to Dramatic Effects of EP Electroporation (EP) produces substantially more changes in gene expression than SQZ in T cells and results in more downstream activation characteristics. (A) Volcano plots depicting log2 fold change and -log p value for all genes arrayed. Genes in the red squares were plotted in (B) a heat map corresponding to >2-fold difference and p < 0.05 and submitted to (C) pathway analysis, both depicting large changes in EP. (D) qPCR confirmed altered gene expression of key activation markers IFNγ and IL-2 in electroporated samples with an without inclusion of Cas9 ribonucleoprotein (RNP) complexes. (E) Cytokine and chemokine secretion of over 24 hours post-treatment were assayed by multiplex ELISA in 3 donors, with greater levels of cytokines secreted in response to EP. (F) Selected cytokines elevated in EP but not SQZ by multiplex ELISA analysis. (**, p <0.01; *, p <0.05 compared to untreated) BA
- Sharei A, et al. "A vector-free microfluidic platform for intracellular delivery." PNAS, 2013 Feb
5;110(6):2082-7
- Zhang M, et al. “The impact of Nucleofection® on the activation state of primary human CD4 T cells.” J
Immunol Methods, 2014 Jun;408:123-131.
Introduction
SQZ Platform Comparable Delivery & Viability Efficiencies Observed Between SQZ and EP Evaluation of delivery and editing efficiencies resulting from electroporation (EP) and SQZ techniques in T cells. (A) Human T cells were isolated and subjected to cargo-free SQZ or EP. (B) Viability and delivery were optimized over an array of EP programs and SQZ pressures, with best-performing conditions underlined and used hereon.
Conclusions
- Squeezed cells have fewer off-target effects and minimal changes in gene
expression compared to electroporation.
- SQZ maintains normal cell phenotypes, whereas electroporation (EP) results in
large changes in gene expression which persist for at least 24 hours.
- SQZ-edited cells maintain a proliferative capacity and cytokine production
which more closely resemble untreated cells, while electroporated cells exhibit poor proliferative capacity and cytokine production changes that persist for > 1 week, suggesting potential exhaustion of electroporated cells.
- SQZ-mediated delivery of CRISPR-Cas9 results in targeted sequence disruption
at multiple genes which correspond to significant reductions in target surface protein expression.
- SQZ enables AAV capsid delivery with similar efficiency to transduction in T
cells and enhanced transgene expression in NK cells, suggesting SQZ can deliver large molecules to the cell and achieve transgene expression in cell types which are less amenable to transduction.
- SQZ is capable of co-delivering RNPs targeting multiple genes with no loss in
cutting frequency and maintained surface protein knock-down of all three targets within the same cell.
- Cell Squeeze® enables effective genome engineering, achieves specific gene
editing, and minimizes disruption of cellular function. Cell Squeezing Enables Effective Genome Engineering and Minimizes Disruption of Cellular Function Contact Information Jacquelyn Hanson, PhD SQZ Biotechnologies, jacquelyn.hanson@sqzbiotech.com B
References
C Cytokines/chemokines → E
Epsqz
D Ifnγ Il-2 F IFNγIL-2 IL-9
Mapre3
Tspan
Emp1
Lmna1
Cd109
Cd200
Ifng
Cxcl9
Il2
A Ep Sqz
Squeezing AAV Results in Efficient Transgene Expression in T & NK Cells 100,000 MOI 900,000 MOID a y 3 D a y 5 D a y 7 D a y 3 D a y 5 D a y 7 2 0 4 0 6 0 8 0 1 0 0 A A V - C M V - G F P E x p r e s s io n in T c e lls
% G F P +
N C
T ra n s d u c tio n S Q ZD a y 2 D a y 4 D a y 6 D a y 8 2 0 4 0 6 0 N K c e lls + A A V -C M V -G F P
% G F P +
T d
S Q Z
Squeezing AAV results in greater transgene expression in NK cells compared to transduction and comparable expression in T cells. (A) Unstimulated human T cells squeezed with AAV capsid expressed GFP transgene comparably to transduced cells. T cells were activated postdelivery to stimulate transgene expression. (B) Unstimulated human NK cells squeezed with AAV capsid expressed a greater amount of GFP transgene compared to transduced NK cells. Transgene expression followed NK cell stimulation against acute myeloid leukemia (AML) cells. BAgB2M-3 gB2M-4 gB2M-6
% B2m K/o
B2M Editing by T7 High CRISPR/Cas9 Editing Efficiencies Observed Across Multiple Genes Using SQZ B CRISPR/Cas9 editing evaluation using SQZ across multiple loci. (A) PD-1 surface knock-down in human T cells (3 donors) was comparable between SQZ and EP, and (B) representative PD-1 knockout efficiency as measured by T7 assay was also comparable. At least 50% surface knock-down in human T cells was achieved across several guides delivered via SQZ at the (C) TRAC and (D) B2M loci with (E & F) coordinating T7 genomic editing estimation. Surface knock-down values calculated as percent protein positive by FACS in RNP-delivered sample compared to untreated. (**, p <0.01; *, p <0.05 compared to no guide)
Asqz Ep
PD-1 Surface Knock-down
% Pd-1 K/d Dc
F Eg3 g5 g7 g8 TRAC Editing by T7 Analysis % TCRa/b K/O Efficient Multiplex Editing Using SQZ DeliveryB2M PD-1 TRAC % Editing by T7 Analysis (n=3) Target Gene % Digest Band Single RNP Multiplex RNP
A B
Multiplex-squeezed CRISPR/Cas9 RNPs facilitate multiple edits per cell and maintains editing efficiency at the single-guide level. (A) Surface knock-down in activated human T cells was comparable between single-target squeezes and combined RNP squeezes for three genes, with (B) confirmation by T7 genomic analysis. (**, p <0.01; *, p <0.05 compared to SQZ alone) (C) In a representative donor, cells gated on B2M K/D population exhibited greater PD-1 and TRAC K/D compared to the unedited B2M population, suggesting multiple edits on a per-cell basis. Csqueeze electroporation Fold change (rel to control)5.0 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 -5 % positive 1 Cells and target cargo together in suspension Cells are squeezed through SQZ chip at high speeds Cell membranes are temporarily disrupted Target cargo is inserted into the cytosol of the cells Membranes reseal Human T Cell Proliferation is Unaffected by SQZ Compared to Slowed Growth Post-EP Assessment of cell state and proliferative capacity in activated human T cells after delivery of gene editing materials using SQZ compared to electroporation (EP). (A) Activated human T cells squeezed with gene editing materials and cultured under stimulating conditions proliferated at a similar rate to untreated, compared to electroporated cells which reduced in number until day 3. Cell numbers determined using FACS-calculated live cell counts. (**, p <0.01; *, p <0.05 compared to untreated) (B) Cytokine production in squeezed cells more closely resembled untreated cells compared to electroporated cells which maintained high levels of cytokine production through Day 10 post-delivery. Quantification by intracellular staining and FACS analysis. A BDay 2 Day 10 IFNy 15Day 2 Day 10
Il-2
80Day 2 Day 10 TNFa 25Day 2 Day 10
Il-2
80Day 2 Day 10 IFNy 15Day 2 Day 10 TNFa 25T cell Proliferation Cytokine Production0 1×104 1×105 1×106 1×107 1×108 1×109 1×1010 T cell Proliferation Day Cell count * ***
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.
# SQZ microfluidic platform enables delivery of gene editing materials and transduction of difficult to engineer cell types while preserving cell phenotype and function
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: SQZ microfluidic platform enables delivery of gene editing materials and transduction of difficult to engineer cell types while preserving cell phenotype and function
- Document type: Poster
- Year: 2020
- Outlet: KEYSTONE SYMPOSIA: ENGINEERING THE GENOME
- Cells: CD34+, T cells, NK cells
- Cargos: Polymers, Viral Particle
- Page URL: https://portal.bio/publications/2020-sqz-microfluidic-platform-enables-delivery-of-gene-editing-materials-and-transduction--ajkHEhEAAC4Ac-B1
- Markdown URL: https://portal.bio/publications/2020-sqz-microfluidic-platform-enables-delivery-of-gene-editing-materials-and-transduction--ajkHEhEAAC4Ac-B1.md
- Original file: https://portal-bio.cdn.prismic.io/portal-bio/65ccfe759be9a5b998b5d300_Keystone-Engineering-the-Genome-2020-Poster-J.-Hanson.pdf
## Summary
Jacquelyn Hanson, Emily McNally, Kendra Foley, Patricia Cho, Joshua Bugge, Tia DiTommaso, Jonathan Gilbert, Marija Tadin-Strapps, Howard Bernstein, and Armon Sharei
## Publication text
no guide gB2M-3 gB2M-4 gB2M-6 % B2M positive B2M Surface Expression ** * **no guide g3 g5 g7 g8 TRAC Surface Expression % TCRa/b positive * *SQZ Microfluidic Platform Enables Delivery of Gene Editing Materials and Transduction of Difficult to Engineer Cell Types While Preserving Cell Phenotype and Function Jacquelyn Hanson, Emily McNally, Kendra Foley, Patricia Cho, Joshua Bugge, Tia DiTommaso, Jonathan Gilbert, Marija Tadin-Strapps, Howard Bernstein, and Armon Sharei SQZ Biotechnologies, Watertown, MA, USA Cell Squeeze® Enables Efficient Primary Immune Cell Editing with Preserved Function
- The translational potential of cell-based therapies is often limited by
complications related to effectively engineering and manufacturing functional cells, including low efficiency, cytotoxicity, off-target effects, and limited scalability.
- Specifically, Zhang et al found that EP treatment of primary T cells
substantially alters cell state, including increases in intracellular calcium levels, increased T cell activation markers CD154 and CD69, and overall increases in aberrant transcriptional activity. (2)
- By using mechanical deformation via Cell Squeeze®, we can achieve
efficient genome editing with minimal perturbation of gene expression or cytokine secretion, and preserved T cell function in vitro. Cell Engineering Capabilities Enabled by the Cell Squeeze® Platform
- Our microfluidic approach to delivery, Cell Squeeze® (SQZ), mechanically
deforms cell membranes through constricting channels, resulting in payload diffusion from the surrounding buffer directly into the cytosol. (1)
- SQZ reduces cell toxicity and off-target effects observed with conventional
delivery methods by eliminating the need for electrical fields or exogenous materials such as viral vectors. These advantages differentiate SQZ as a potentially transformative platform for enabling cell therapies that have been historically challenging to engineer, including the use of CRISPR to engineer cell function.
- Cells subjected to SQZ have minimal aberrant transcriptional responses
compared to electroporation (EP) and exhibit editing efficiencies comparable to conventional methods such as EP.
- SQZ-mediated engineering of immune cells results in the desired
phenotype with minimal effect on viability, baseline gene expression, and cell function compared to EP. SQZ 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. Minimal Side Effects With SQZ Platform in Contrast to Dramatic Effects of EP Electroporation (EP) produces substantially more changes in gene expression than SQZ in T cells and results in more downstream activation characteristics. (A) Volcano plots depicting log2 fold change and -log p value for all genes arrayed. Genes in the red squares were plotted in (B) a heat map corresponding to >2-fold difference and p < 0.05 and submitted to (C) pathway analysis, both depicting large changes in EP. (D) qPCR confirmed altered gene expression of key activation markers IFNγ and IL-2 in electroporated samples with an without inclusion of Cas9 ribonucleoprotein (RNP) complexes. (E) Cytokine and chemokine secretion of over 24 hours post-treatment were assayed by multiplex ELISA in 3 donors, with greater levels of cytokines secreted in response to EP. (F) Selected cytokines elevated in EP but not SQZ by multiplex ELISA analysis. (**, p <0.01; *, p <0.05 compared to untreated) BA
1. Sharei A, et al. "A vector-free microfluidic platform for intracellular delivery." PNAS, 2013 Feb
5;110(6):2082-7
1. Zhang M, et al. “The impact of Nucleofection® on the activation state of primary human CD4 T cells.” J
Immunol Methods, 2014 Jun;408:123-131.
### Introduction
SQZ Platform Comparable Delivery & Viability Efficiencies Observed Between SQZ and EP Evaluation of delivery and editing efficiencies resulting from electroporation (EP) and SQZ techniques in T cells. (A) Human T cells were isolated and subjected to cargo-free SQZ or EP. (B) Viability and delivery were optimized over an array of EP programs and SQZ pressures, with best-performing conditions underlined and used hereon.
### Conclusions
- Squeezed cells have fewer off-target effects and minimal changes in gene
expression compared to electroporation.
- SQZ maintains normal cell phenotypes, whereas electroporation (EP) results in
large changes in gene expression which persist for at least 24 hours.
- SQZ-edited cells maintain a proliferative capacity and cytokine production
which more closely resemble untreated cells, while electroporated cells exhibit poor proliferative capacity and cytokine production changes that persist for > 1 week, suggesting potential exhaustion of electroporated cells.
- SQZ-mediated delivery of CRISPR-Cas9 results in targeted sequence disruption
at multiple genes which correspond to significant reductions in target surface protein expression.
- SQZ enables AAV capsid delivery with similar efficiency to transduction in T
cells and enhanced transgene expression in NK cells, suggesting SQZ can deliver large molecules to the cell and achieve transgene expression in cell types which are less amenable to transduction.
- SQZ is capable of co-delivering RNPs targeting multiple genes with no loss in
cutting frequency and maintained surface protein knock-down of all three targets within the same cell.
- Cell Squeeze® enables effective genome engineering, achieves specific gene
editing, and minimizes disruption of cellular function. Cell Squeezing Enables Effective Genome Engineering and Minimizes Disruption of Cellular Function Contact Information Jacquelyn Hanson, PhD SQZ Biotechnologies, jacquelyn.hanson@sqzbiotech.com B
### References
C Cytokines/chemokines → E
### Epsqz
D Ifnγ Il-2 F IFNγIL-2 IL-9
### Mapre3
### Tspan
### Emp1
### Lmna1
### Cd109
### Cd200
### Ifng
### Cxcl9
### Il2
### A Ep Sqz
Squeezing AAV Results in Efficient Transgene Expression in T & NK Cells 100,000 MOI 900,000 MOID a y 3 D a y 5 D a y 7 D a y 3 D a y 5 D a y 7 2 0 4 0 6 0 8 0 1 0 0 A A V - C M V - G F P E x p r e s s io n in T c e lls
### % G F P +
### N C
T ra n s d u c tio n S Q ZD a y 2 D a y 4 D a y 6 D a y 8 2 0 4 0 6 0 N K c e lls + A A V -C M V -G F P
### % G F P +
T d
### S Q Z
Squeezing AAV results in greater transgene expression in NK cells compared to transduction and comparable expression in T cells. (A) Unstimulated human T cells squeezed with AAV capsid expressed GFP transgene comparably to transduced cells. T cells were activated postdelivery to stimulate transgene expression. (B) Unstimulated human NK cells squeezed with AAV capsid expressed a greater amount of GFP transgene compared to transduced NK cells. Transgene expression followed NK cell stimulation against acute myeloid leukemia (AML) cells. BAgB2M-3 gB2M-4 gB2M-6
### % B2m K/o
B2M Editing by T7 High CRISPR/Cas9 Editing Efficiencies Observed Across Multiple Genes Using SQZ B CRISPR/Cas9 editing evaluation using SQZ across multiple loci. (A) PD-1 surface knock-down in human T cells (3 donors) was comparable between SQZ and EP, and (B) representative PD-1 knockout efficiency as measured by T7 assay was also comparable. At least 50% surface knock-down in human T cells was achieved across several guides delivered via SQZ at the (C) TRAC and (D) B2M loci with (E & F) coordinating T7 genomic editing estimation. Surface knock-down values calculated as percent protein positive by FACS in RNP-delivered sample compared to untreated. (**, p <0.01; *, p <0.05 compared to no guide)
### Asqz Ep
PD-1 Surface Knock-down
### % Pd-1 K/d Dc
F Eg3 g5 g7 g8 TRAC Editing by T7 Analysis % TCRa/b K/O Efficient Multiplex Editing Using SQZ DeliveryB2M PD-1 TRAC % Editing by T7 Analysis (n=3) Target Gene % Digest Band Single RNP Multiplex RNP
### A B
Multiplex-squeezed CRISPR/Cas9 RNPs facilitate multiple edits per cell and maintains editing efficiency at the single-guide level. (A) Surface knock-down in activated human T cells was comparable between single-target squeezes and combined RNP squeezes for three genes, with (B) confirmation by T7 genomic analysis. (**, p <0.01; *, p <0.05 compared to SQZ alone) (C) In a representative donor, cells gated on B2M K/D population exhibited greater PD-1 and TRAC K/D compared to the unedited B2M population, suggesting multiple edits on a per-cell basis. Csqueeze electroporation Fold change (rel to control)5.0 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 -5 % positive 1 Cells and target cargo together in suspension Cells are squeezed through SQZ chip at high speeds Cell membranes are temporarily disrupted Target cargo is inserted into the cytosol of the cells Membranes reseal Human T Cell Proliferation is Unaffected by SQZ Compared to Slowed Growth Post-EP Assessment of cell state and proliferative capacity in activated human T cells after delivery of gene editing materials using SQZ compared to electroporation (EP). (A) Activated human T cells squeezed with gene editing materials and cultured under stimulating conditions proliferated at a similar rate to untreated, compared to electroporated cells which reduced in number until day 3. Cell numbers determined using FACS-calculated live cell counts. (**, p <0.01; *, p <0.05 compared to untreated) (B) Cytokine production in squeezed cells more closely resembled untreated cells compared to electroporated cells which maintained high levels of cytokine production through Day 10 post-delivery. Quantification by intracellular staining and FACS analysis. A BDay 2 Day 10 IFNy 15Day 2 Day 10
### Il-2
80Day 2 Day 10 TNFa 25Day 2 Day 10
### Il-2
80Day 2 Day 10 IFNy 15Day 2 Day 10 TNFa 25T cell Proliferation Cytokine Production0 1×104 1×105 1×106 1×107 1×108 1×109 1×1010 T cell Proliferation Day Cell count * ***
## 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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