Poster · 2024 · ISCT: International Society for Cell & Gene Therapy

A platform for RNA-based and genomic cell engineering: simplifying multiplexed delivery to human primary immune and stem cells

Zhihui Song, Sophia Hirsch, Rachel Conover, Andrew Laroque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei

Cells: T cells, iPSCs, PBMCs, CD34+, Cancer Cell Lines
Cargos: mRNA, CRISPR RNPs, siRNA, Antibody, Oligo

Publication text

Dextran PBMCs Robust mRNA expression in a variety of immune cells GFP mRNA Boost

(cd3+)

B cells

(cd19+)

NK cells

(cd56+)

Monocytes

(cd14+)

Gfp

Peripheral blood mononucleated cell (PBMC) boost with mRNA High Knock Down Efficiency in CD34+ Cells Hematopoietic Stem Cell (HSC) boost with siRNA siRNA CD34+ cells Efficient mRNA Expression in Human iPSCs Induced pluripotent stem cell (iPSC) boost with GFP mRNA GFP mRNA GFP mRNA was mechanoporated into peripheral blood mononuclear cells (PBMCs). Flow cytometry was conducted 20h post-boost to assess viability, GFP expression, and delivery to different cell types. β2 microglobulin (B2M) siRNA was mechanoporated into human CD34+ hematopoietic stem cells. Flow cytometry was conducted after 40 hours to assess viability and B2M expression. iPSCs Induced pluripotent stem cells (iPSCs) were mechanoporated with GFP mRNA. After 20h, GFP expression was assessed using fluorescence microscopy and flow cytometry. Future directions: Using Portal’s Mechanoporation Technology for Eliminating Preconditioning Mutant BCL2 enables resistance to Venetoclax-induced apoptosis and promotes HSC survival No Treatment Venetoclax

Gfp Bcl2

G101v

Transferred cell frequency (% of human cells) Boost HSCs to increase homing and cell survival towards reducing pre-conditioning for cell therapies CD34+ cells were mechanoporated with either GFP or BCL-2 (G101V) mRNA and transferred to NSG mice with or without Venetoclax treatment. Bone marrow was harvested and frequency of transplanted HSCs was measured. Silva et al., ASH 2022 Silver et al., ESMO 2022 mRNA encoded membrane bound (mb) IL-2/12 cytokines were boosted into T cells and implanted into mice. Blood was drawn after 5 days and % of T cells expressing mbIL-2/12 was measured, illustrating potential to improve memory phenotype and cytotoxicity in a mouse model. TCM-like TEM-like TEFF-like Boost immune cells to improve effector function for cell therapy

Summary

● Portal’s mechanoporation technology can be used for RNA-based cell engineering ○ Compatible across broad cell types including: ○ Can be used to deliver single RNAs or multiplexed cargoes ● Portal’s Research Scale Gateway™ enables complex cell engineering with a simple workflow ● Little impact on normal cellular function ● Portal plans to use its technology for transient modifications of cells for: ○ Cell therapies ○ Biomanufacturing ○ Regenerative medicine ○ Diagnostics and Analysis ○ High throughput screening GFP Transmitted light Boost Untreated Research-Scale System Gateway™ Consumable Delivery Cartridge Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet MicroBooster™ Defined pore size for deformation Cell type-specific consumable for optimal mechanoporationmediated cargo delivery 18mm 35mm Introduction to Portal’s Approach: Implementing a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular Delivery Memory Survival Killing HomingmRNA siRNA Cells and Cargo Cells Cargo ● Portal is advancing a transformative technology for cellular engineering based on mechanical deformation that allows cytosolic delivery of a multitude of cargoes including mRNA, CRISPR RNPs, peptides, DELs and many others1-4 ● The technology is compatible with many primary cell types as well as cell lines including immune and stem cells ● Cells are engineered (‘boosted’) through a membrane at low operating pressure (3-15 PSI) ● Membrane format5 enables high-throughput cellular engineering

Gfp Bcl2

G101v

A platform for RNA-based and genomic cell engineering: simplifying multiplexed delivery to human primary immune and stem cells Zhihui Song, Sophia Hirsch, Rachel Conover, Andrew Laroque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA Robust Multiplex Delivery in Unstimulated T Cells T cell boost with CRISPR RNP, mRNA and Dextran GFP expression Dextran, mRNA, &

B2m Crispr Rnp

Unstimulated T cells Unstimulated T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNP. After 40h, expression of mRNA and B2M protein were assessed by flow cytometry. Efficient Delivery of mRNA to a variety of immune cell types (1) Sharei et al., PNAS, 2013 (2) Szeto et al., Scientific Reports, 2015 (3) Sharei et al., PLOS One, 2015 (4) Sharei et al., Integrative Biology, 2014 (5) Williams AR, Bao S, Miller DL, Biotechnol Bioeng., 1999 Connect with Portal! Future Directions: Using Portal’s Mechanoporation Technology for Transient Enhancement of Cell Function

Toda

Future

By engineering the cell, not the patient we can increase safety, reduce costs, and expand access Toxic chemotherapy pre-conditioning can lead to:

Death, infertility, and risk of infection
Often a week or more in hospitalization

Cells engraft with no or mild preconditioning Toxicity, side effects, hospitalizations Cells engraft in immune depleted patient

Engraftmentpre-conditioning

Contact us for beta program eligibility info@portal.bio ● Low pressure requirements enable integration into common manufacturing equipment ● “Powered by Portal” consumables Clinical / Process Scale Integration with 3rd party cellular manufacturing Activation and expansion

Trac & B2m

CRISPR RNPs Multiplexed CRISPR KO in Unstimulated T Cells TRAC expression Unstimulated T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing CRISPR KO RNPs against T Cell Receptor Alpha Constant (TRAC) and β2 microglobulin (B2M). T cells were activated and expanded for 4 days and expression of TRAC protein, B2M protein were assessed by flow cytometry.

B2m

Crispr

Rnp

Activated T cells Unstimulated T cells were mechanoporated with β2 microglobulin (B2M) CRISPR RNP. T cells were activated and expanded in G-Rex plates for 9 days. Then, dextran polymers were mechanoporated at clinical scale. Flow cytometry was conducted post-boost to assess B2M protein expression and efficiency of dextran delivery. Robust Sequential Delivery at Clinical Scale Dextran Unstimulated T cell boost with multiplexed RNPs followed by activation Unstimulated T cell boost followed by activation & boost at clinical scale Activation and expansion Viability of Activated T Cells NK cells HSCs B cellsPBMCs T cells iPSCs HeLa

Hek

Efficient Co-delivery of Antibody, Oligos, and Dextran to HeLa Cells Mixed isotype control antibody, labeled oligo, and fluorescent dextran HeLa cells Antibody Oligo Dextran Fluorescently-labeled IgG isotype control antibody, Cy5-labeled oligo, and fluorescent dextran were mixed and mechanoporated into HeLa cells. Flow cytometry was conducted post-boost to assess viability and delivery of the cargo types to the cells.

B2m+

Trac+

B2m-

Trac-

Download PDF

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.

# A platform for RNA-based and genomic cell engineering: simplifying multiplexed delivery to human primary immune and stem cells 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: A platform for RNA-based and genomic cell engineering: simplifying multiplexed delivery to human primary immune and stem cells - Document type: Poster - Year: 2024 - Outlet: ISCT: International Society for Cell & Gene Therapy - Cells: T cells, iPSCs, PBMCs, CD34+, Cancer Cell Lines - Cargos: mRNA, CRISPR RNPs, siRNA, Antibody, Oligo - Page URL: https://portal.bio/publications/2024-a-platform-for-rna-based-and-genomic-cell-engineering-simplifying-multiplexed-deliver--ajkGWxEAAC4Ac94O - Markdown URL: https://portal.bio/publications/2024-a-platform-for-rna-based-and-genomic-cell-engineering-simplifying-multiplexed-deliver--ajkGWxEAAC4Ac94O.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFmk-3fc4bHWioab_PosterISCT2024_v1.pdf ## Summary Zhihui Song, Sophia Hirsch, Rachel Conover, Andrew Laroque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei ## Publication text Dextran PBMCs Robust mRNA expression in a variety of immune cells GFP mRNA Boost ### (cd3+) B cells ### (cd19+) NK cells ### (cd56+) Monocytes ### (cd14+) ### Gfp Peripheral blood mononucleated cell (PBMC) boost with mRNA High Knock Down Efficiency in CD34+ Cells Hematopoietic Stem Cell (HSC) boost with siRNA siRNA CD34+ cells Efficient mRNA Expression in Human iPSCs Induced pluripotent stem cell (iPSC) boost with GFP mRNA GFP mRNA GFP mRNA was mechanoporated into peripheral blood mononuclear cells (PBMCs). Flow cytometry was conducted 20h post-boost to assess viability, GFP expression, and delivery to different cell types. β2 microglobulin (B2M) siRNA was mechanoporated into human CD34+ hematopoietic stem cells. Flow cytometry was conducted after 40 hours to assess viability and B2M expression. iPSCs Induced pluripotent stem cells (iPSCs) were mechanoporated with GFP mRNA. After 20h, GFP expression was assessed using fluorescence microscopy and flow cytometry. Future directions: Using Portal’s Mechanoporation Technology for Eliminating Preconditioning Mutant BCL2 enables resistance to Venetoclax-induced apoptosis and promotes HSC survival No Treatment Venetoclax ### Gfp Bcl2 ### G101v Transferred cell frequency (% of human cells) Boost HSCs to increase homing and cell survival towards reducing pre-conditioning for cell therapies CD34+ cells were mechanoporated with either GFP or BCL-2 (G101V) mRNA and transferred to NSG mice with or without Venetoclax treatment. Bone marrow was harvested and frequency of transplanted HSCs was measured. Silva et al., ASH 2022 Silver et al., ESMO 2022 mRNA encoded membrane bound (mb) IL-2/12 cytokines were boosted into T cells and implanted into mice. Blood was drawn after 5 days and % of T cells expressing mbIL-2/12 was measured, illustrating potential to improve memory phenotype and cytotoxicity in a mouse model. TCM-like TEM-like TEFF-like Boost immune cells to improve effector function for cell therapy ### Summary ● Portal’s mechanoporation technology can be used for RNA-based cell engineering ○ Compatible across broad cell types including: ○ Can be used to deliver single RNAs or multiplexed cargoes ● Portal’s Research Scale Gateway™ enables complex cell engineering with a simple workflow ● Little impact on normal cellular function ● Portal plans to use its technology for transient modifications of cells for: ○ Cell therapies ○ Biomanufacturing ○ Regenerative medicine ○ Diagnostics and Analysis ○ High throughput screening GFP Transmitted light Boost Untreated Research-Scale System Gateway™ Consumable Delivery Cartridge Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet MicroBooster™ Defined pore size for deformation Cell type-specific consumable for optimal mechanoporationmediated cargo delivery 18mm 35mm Introduction to Portal’s Approach: Implementing a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular Delivery Memory Survival Killing HomingmRNA siRNA Cells and Cargo Cells Cargo ● Portal is advancing a transformative technology for cellular engineering based on mechanical deformation that allows cytosolic delivery of a multitude of cargoes including mRNA, CRISPR RNPs, peptides, DELs and many others1-4 ● The technology is compatible with many primary cell types as well as cell lines including immune and stem cells ● Cells are engineered (‘boosted’) through a membrane at low operating pressure (3-15 PSI) ● Membrane format5 enables high-throughput cellular engineering ### Gfp Bcl2 ### G101v A platform for RNA-based and genomic cell engineering: simplifying multiplexed delivery to human primary immune and stem cells Zhihui Song, Sophia Hirsch, Rachel Conover, Andrew Laroque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA Robust Multiplex Delivery in Unstimulated T Cells T cell boost with CRISPR RNP, mRNA and Dextran GFP expression Dextran, mRNA, & ### B2m Crispr Rnp Unstimulated T cells Unstimulated T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNP. After 40h, expression of mRNA and B2M protein were assessed by flow cytometry. Efficient Delivery of mRNA to a variety of immune cell types (1) Sharei et al., PNAS, 2013 (2) Szeto et al., Scientific Reports, 2015 (3) Sharei et al., PLOS One, 2015 (4) Sharei et al., Integrative Biology, 2014 (5) Williams AR, Bao S, Miller DL, Biotechnol Bioeng., 1999 Connect with Portal! Future Directions: Using Portal’s Mechanoporation Technology for Transient Enhancement of Cell Function ### Toda Y ### Future By engineering the cell, not the patient we can increase safety, reduce costs, and expand access Toxic chemotherapy pre-conditioning can lead to: - Death, infertility, and risk of infection - Often a week or more in hospitalization Cells engraft with no or mild preconditioning Toxicity, side effects, hospitalizations Cells engraft in immune depleted patient ### Engraftmentpre-conditioning Contact us for beta program eligibility info@portal.bio ● Low pressure requirements enable integration into common manufacturing equipment ● “Powered by Portal” consumables Clinical / Process Scale Integration with 3rd party cellular manufacturing Activation and expansion ### Trac & B2m CRISPR RNPs Multiplexed CRISPR KO in Unstimulated T Cells TRAC expression Unstimulated T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing CRISPR KO RNPs against T Cell Receptor Alpha Constant (TRAC) and β2 microglobulin (B2M). T cells were activated and expanded for 4 days and expression of TRAC protein, B2M protein were assessed by flow cytometry. ### B2m ### Crispr ### Rnp Activated T cells Unstimulated T cells were mechanoporated with β2 microglobulin (B2M) CRISPR RNP. T cells were activated and expanded in G-Rex plates for 9 days. Then, dextran polymers were mechanoporated at clinical scale. Flow cytometry was conducted post-boost to assess B2M protein expression and efficiency of dextran delivery. Robust Sequential Delivery at Clinical Scale Dextran Unstimulated T cell boost with multiplexed RNPs followed by activation Unstimulated T cell boost followed by activation & boost at clinical scale Activation and expansion Viability of Activated T Cells NK cells HSCs B cellsPBMCs T cells iPSCs HeLa ### Hek Efficient Co-delivery of Antibody, Oligos, and Dextran to HeLa Cells Mixed isotype control antibody, labeled oligo, and fluorescent dextran HeLa cells Antibody Oligo Dextran Fluorescently-labeled IgG isotype control antibody, Cy5-labeled oligo, and fluorescent dextran were mixed and mechanoporated into HeLa cells. Flow cytometry was conducted post-boost to assess viability and delivery of the cargo types to the cells. ### B2m+ ### Trac+ ### B2m- ### Trac- ## 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.