Poster · 2024 · Cell and Gene Therapy Resesach at MGB

A Universal Platform for Multiplexed RNA and Genomic Cell Engineering

Zhihui Song, Eleni Rogers, Sophia Hirsch, Darby Kreienberg, Nicholas Willis, Rachel Conover, Andrew Larocque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Robert Wesselhoeft, Armon Sharei

Cells: PBMCs, T cells, iPSCs
Cargos: mRNA, circRNA, CRISPR RNPs

Publication text

PBMCs GFP mRNA Boost T cells

(cd3+)

B cells

(cd19+)

NK cells

(cd56+)

Monocytes

(cd14+)

Gfp

Peripheral blood mononucleated cell (PBMC) boost with 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. 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 Research-Scale System Gateway™ System Consumable Cartridge Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet Booster™ Cartridges Defined pore size for deformation Cell type-specific consumable for optimal mechanoporationmediated cargo delivery 18mm 35mm Mechanical Membrane Disruption as 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 silicon membrane at low operating pressure (3-15 PSI) ● Membrane format5 enables high-throughput cellular engineering

Gfp Bcl2

G101v

A Universal Platform for Multiplexed RNA and Genomic Cell Engineering Zhihui Song1, Eleni Rogers1, Sophia Hirsch1, Darby Kreienberg1, Nicholas Willis2, Rachel Conover1, Andrew Larocque1, Jacquelyn Hanson1, Alec Barclay1, Mathias Pawlak2, Robert Wesselhoeft1, Armon Sharei1 1 Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA 2 Mass General Brigham Gene and Cell Therapy Institute，65 Landsdowne St., Cambridge, MA 02139, 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 Sharei, et al., PNAS, 2013, Szeto, et al., Scientific Reports, 2015, Sharei, et al., PLOS One, 2015, Sharei et al., Integrative Biology 2014, 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 Robust mRNA expression in a variety of immune cells Activation and expansion T cells

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. Unstimulated T cell boost with multiplexed RNPs followed by activation Viability of Activated T Cells NK cells HSCs B cellsPBMCs T cells iPSCs HeLa

Hek

Activated T cells Engineered with Simultaneous circRNA Delivery for membrane bound IL-12 and CD19 CAR Pan-T cells were activated with Dynabeads, and the beads were removed after 48 hours of activation. After 4 days of culturing, the activated T cells were mechanoporated with circRNA encoding CD19 CAR and mbIL-12. After a 2-hour rest, the boosted cells were co-cultured with Raji cells at different ratios. After 2 days of co-culture, Raji cells were counted using counting beads. Activated T cells

(d6)

CD19 CAR cRNA mbIL12-cRNA

Cd19 Car

Il12

Gfp

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, GFP mRNA were mechanoporated at clinical scale. Flow cytometry was conducted 48h post-boost to assess B2M protein expression and efficiency of GFP expression. Robust Sequential Delivery at Clinical Scale Unstimulated T cell boost followed by activation & clinical scale boost 3 kDa Dextran Dextran delivery Ngn2 Human stem cells (iPSCs) Human stem-cell derived neurons iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA 3 kDa dextran and Ngn2 mRNA were mechanoporated into human iPSCs. mRNA was extracted 12h post-boost and RT-qPCR was performed to amplify neuronal-specific mRNA expressed downstream of Ngn2. T Cells Boost Gateway Activation and Expansion Edited, Activated T CellsLovo + Clinical Scale BoosterRNA expressing and edited T Cells Robust T cell expansion, viability, editing and mRNA expression post-boost Cell Expansion Viable Cell Count (M) 5 million/well at onset Viability B2M KO efficiency(%) mRNA Expression B2M KO (%)GFP expression (%) Viability (%)

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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 Universal Platform for Multiplexed RNA and Genomic Cell Engineering 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 Universal Platform for Multiplexed RNA and Genomic Cell Engineering - Document type: Poster - Year: 2024 - Outlet: Cell and Gene Therapy Resesach at MGB - Cells: PBMCs, T cells, iPSCs - Cargos: mRNA, circRNA, CRISPR RNPs - Page URL: https://portal.bio/publications/2024-a-universal-platform-for-multiplexed-rna-and-genomic-cell-engineering--ajkGPxEAAC0Ac92r - Markdown URL: https://portal.bio/publications/2024-a-universal-platform-for-multiplexed-rna-and-genomic-cell-engineering--ajkGPxEAAC0Ac92r.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFmtcHfc4bHWiocI_MGB2024_ZhihuiSong.pdf ## Summary Zhihui Song, Eleni Rogers, Sophia Hirsch, Darby Kreienberg, Nicholas Willis, Rachel Conover, Andrew Larocque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Robert Wesselhoeft, Armon Sharei ## Publication text PBMCs GFP mRNA Boost T cells ### (cd3+) B cells ### (cd19+) NK cells ### (cd56+) Monocytes ### (cd14+) ### Gfp Peripheral blood mononucleated cell (PBMC) boost with 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. 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 Research-Scale System Gateway™ System Consumable Cartridge Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet Booster™ Cartridges Defined pore size for deformation Cell type-specific consumable for optimal mechanoporationmediated cargo delivery 18mm 35mm Mechanical Membrane Disruption as 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 silicon membrane at low operating pressure (3-15 PSI) ● Membrane format5 enables high-throughput cellular engineering ### Gfp Bcl2 ### G101v A Universal Platform for Multiplexed RNA and Genomic Cell Engineering Zhihui Song1, Eleni Rogers1, Sophia Hirsch1, Darby Kreienberg1, Nicholas Willis2, Rachel Conover1, Andrew Larocque1, Jacquelyn Hanson1, Alec Barclay1, Mathias Pawlak2, Robert Wesselhoeft1, Armon Sharei1 1 Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA 2 Mass General Brigham Gene and Cell Therapy Institute，65 Landsdowne St., Cambridge, MA 02139, 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 Sharei, et al., PNAS, 2013, Szeto, et al., Scientific Reports, 2015, Sharei, et al., PLOS One, 2015, Sharei et al., Integrative Biology 2014, 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 Robust mRNA expression in a variety of immune cells Activation and expansion T cells ### 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. Unstimulated T cell boost with multiplexed RNPs followed by activation Viability of Activated T Cells NK cells HSCs B cellsPBMCs T cells iPSCs HeLa ### Hek Activated T cells Engineered with Simultaneous circRNA Delivery for membrane bound IL-12 and CD19 CAR Pan-T cells were activated with Dynabeads, and the beads were removed after 48 hours of activation. After 4 days of culturing, the activated T cells were mechanoporated with circRNA encoding CD19 CAR and mbIL-12. After a 2-hour rest, the boosted cells were co-cultured with Raji cells at different ratios. After 2 days of co-culture, Raji cells were counted using counting beads. Activated T cells ### (d6) CD19 CAR cRNA mbIL12-cRNA ### Cd19 Car ### Il12 ### Gfp 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, GFP mRNA were mechanoporated at clinical scale. Flow cytometry was conducted 48h post-boost to assess B2M protein expression and efficiency of GFP expression. Robust Sequential Delivery at Clinical Scale Unstimulated T cell boost followed by activation & clinical scale boost 3 kDa Dextran Dextran delivery Ngn2 Human stem cells (iPSCs) Human stem-cell derived neurons iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA 3 kDa dextran and Ngn2 mRNA were mechanoporated into human iPSCs. mRNA was extracted 12h post-boost and RT-qPCR was performed to amplify neuronal-specific mRNA expressed downstream of Ngn2. T Cells Boost Gateway Activation and Expansion Edited, Activated T CellsLovo + Clinical Scale BoosterRNA expressing and edited T Cells Robust T cell expansion, viability, editing and mRNA expression post-boost Cell Expansion Viable Cell Count (M) 5 million/well at onset Viability B2M KO efficiency(%) mRNA Expression B2M KO (%)GFP expression (%) Viability (%) ## 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.