Poster · 2024 · ESGCT

A platform for multiplexed cell engineering – Silicon membranes for robust mechanical delivery of diverse cargoes to human stem and immune cells

Cells: T cells, iPSCs, PBMCs, NK cells
Cargos: mRNA, siRNA, circRNA

Publication text

A platform for multiplexed cell engineering – Silicon membranes for robust mechanical delivery of diverse cargoes to human stem and immune cells Sophia Hirsch, Zhihui Song, Eleni Rogers, Darby Kreienberg, Rachel Conover, Andrew Laroque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA PBMCs Robust mRNA expression in a variety of immune cells GFP mRNA Boost T cells

(cd3+)

B cells

(cd19+)

NK cells

(cd56+)

Monocytes

(cd14+)

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 B2M expression 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. Efficient Delivery of mRNA to mixed PBMC populations

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 Memory Survival Killing HomingmRNA siRNA Connect with Portal! Contact us for beta program eligibility info@portal.bio NK cells HSCs B cellsPBMCs T cells iPSCs Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Human Pan-T cells were isolated and activated with CD3/CD28 Dynabeads for 48h and T cells were cultured an additional 4 days. Activated T cells were mechanoporated with CD19 CAR circRNA and membrane bound IL-12 (mbIL-12) circRNA. Boosted cells were rested for 2 h before co-culturing with Raji cells at different ratios of effector cells (E) to target cells (T). Flow cytometry was conducted 48h post-boost to assess expression of CD19 CAR and mbIL-12 (left) and cytotoxicity (right). Cytotoxicity was measured by quantifying reduction in CD19+ Raji cells relative to counting beads. Activated T cells

(d6)

CD19 CAR circRNA mblL-12 circRNA Research Scale Gateway™ High Throughput Compact bench-top unit that controls delivery parameters and easily fits in hood Introduction to Portal’s Approach: Implementing A Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo

Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999

● Over a billion cells in under a minute ● “Powered by Portal” consumables Clinical Scale Integrate with 3rd Party manufacturing equipment ● Portal has created a cell engineering technology compatible across cell types and materials ○ Primary and patient-derived cell lines including immune and stem cells ○ mRNA, siRNA, cRNA, saRNA, DNA, RNPs, proteins, and other cargos ● Cells are mechanoporated (“boosted”) through a membrane at low operating pressure (3-15 PSI) ○ Membrane format1 enables high-throughput cellular engineering ● Integrates with existing liquid handlers ● Ready for early adopters Q1 2025 “Powered by Portal” Consumables Multiplex Delivery in Unstimulated T Cells T Cell Boost with CRISPR RNP, mRNA, and Dextran B2M expression Control Boost Mixed dextran, mRNA, & RNP Naive T cells Efficient mRNA Expression in Human iPSCs and MSCs iPSCs and MSCs boosted with GFP mRNA iPSCs Induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs) were mechanoporated with GFP mRNA. After 20h, GFP expression in iPSCs was assessed using fluorescence microscopy and flow cytometry. GFP expression in MSCs was assessed using flow cytometry and cell morphology was assessed by microscopy. Size (microns) refers to pore size used for mechanoporation. GFP expression 10 μm 11 μm GFP Transmitted light Boost Untreated Circular RNA Expression in Mixed PBMCs Day 3 cRNA GFP mRNA GFP cRNA

-or-

Mixed PBMCs were mechanoporated with either GFP mRNA or GFP cRNA (circular RNA). GFP expression was assessed on days 1, 3, 7, and 10 post-mechanoporation by flow cytometry. MSCs Mixed PBMCs Protocol T Cells Boost Gateway Activation and Expansion Edited, Activated T Cells Lovo + Clinical Scale Booster RNA expressing and edited T Cells Robust T cell expansion, viability, editing and post-boost D0 D4 D7 D10 NK boost with mRNA Mechanoporation yields efficient delivery in NK cells Unstimulated T cells were mechanoporated with β2 microglobulin (B2M) CRISPR RNPs. T cells were activated and expanded in G-Rex plates for 9 days. GFP mRNA were mechanoporated into the activated T cells at clinical scale using Portal’s MilliBooster and . Flow cytometry was conducted 48h post-boost to assess B2M knock out efficiency and efficiency of GFP expression. Robust Sequential Delivery at Clinical Scale Unstimulated T cell boost followed by activation & boost at clinical scale Unstimulated human T cells were isolated from PBMCs and boosted (mechanoporated) with a multiplexed cargo containing GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNPs. Expression of GFP mRNA and B2M protein were assessed by flow cytometry 40h post-boost. Cell Expansion Viable Cell Count (M) 5 million/well at onset Viability Viability (%)

B2m Ko

efficiency(%) Expression

B2m Ko (%)

Control Boost Control BoostD4 D7 D10 GFP expression (%) NK cells were isolated from PBMCs and mechanoporated with GFP mRNA. Viability and mRNA expression in CD3- CD56+ cells were assessed by flow cytometry 24h post-boost. NK cells

Cd19 Car

mbIL-12 Control 3 kDa Dextran Dextran delivery Expression of Ngn2 and downstream neuronal transcription factors Human iPSCs Stem-cell derived neurons iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA Boost with Ngn2 mRNA Induced pluripotent stem cells (iPSCs) were mechanoporated with neurogenin 2 (Ngn2) mRNA and dextran. Dextran delivery was assessed immediately post-boost by flow cytometry. Cells were harvested and total RNA was extracted 12h post boost. RT-qPCR was performed to assess expression levels of Ngn2 and neuronal transcription factors NeuroD1 and NeuroD4. Control Boost Control BoostControl Boost

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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 platform for multiplexed cell engineering – Silicon membranes for robust mechanical delivery of diverse cargoes to human stem and immune 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 multiplexed cell engineering – Silicon membranes for robust mechanical delivery of diverse cargoes to human stem and immune cells - Document type: Poster - Year: 2024 - Outlet: ESGCT - Cells: T cells, iPSCs, PBMCs, NK cells - Cargos: mRNA, siRNA, circRNA - Page URL: https://portal.bio/publications/2024-a-platform-for-multiplexed-cell-engineering-silicon-membranes-for-robust-mechanical-d--ajkGSREAACsAc93Q - Markdown URL: https://portal.bio/publications/2024-a-platform-for-multiplexed-cell-engineering-silicon-membranes-for-robust-mechanical-d--ajkGSREAACsAc93Q.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFotcXfc4bHWiplU_PortalESGCT2024.pdf ## Publication text A platform for multiplexed cell engineering – Silicon membranes for robust mechanical delivery of diverse cargoes to human stem and immune cells Sophia Hirsch, Zhihui Song, Eleni Rogers, Darby Kreienberg, Rachel Conover, Andrew Laroque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA PBMCs Robust mRNA expression in a variety of immune cells GFP mRNA Boost T cells ### (cd3+) B cells ### (cd19+) NK cells ### (cd56+) Monocytes ### (cd14+) 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 B2M expression 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. Efficient Delivery of mRNA to mixed PBMC populations ### 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 Memory Survival Killing HomingmRNA siRNA Connect with Portal! Contact us for beta program eligibility info@portal.bio NK cells HSCs B cellsPBMCs T cells iPSCs Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Human Pan-T cells were isolated and activated with CD3/CD28 Dynabeads for 48h and T cells were cultured an additional 4 days. Activated T cells were mechanoporated with CD19 CAR circRNA and membrane bound IL-12 (mbIL-12) circRNA. Boosted cells were rested for 2 h before co-culturing with Raji cells at different ratios of effector cells (E) to target cells (T). Flow cytometry was conducted 48h post-boost to assess expression of CD19 CAR and mbIL-12 (left) and cytotoxicity (right). Cytotoxicity was measured by quantifying reduction in CD19+ Raji cells relative to counting beads. Activated T cells ### (d6) CD19 CAR circRNA mblL-12 circRNA Research Scale Gateway™ High Throughput Compact bench-top unit that controls delivery parameters and easily fits in hood Introduction to Portal’s Approach: Implementing A Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo 1. Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999 ● Over a billion cells in under a minute ● “Powered by Portal” consumables Clinical Scale Integrate with 3rd Party manufacturing equipment ● Portal has created a cell engineering technology compatible across cell types and materials ○ Primary and patient-derived cell lines including immune and stem cells ○ mRNA, siRNA, cRNA, saRNA, DNA, RNPs, proteins, and other cargos ● Cells are mechanoporated (“boosted”) through a membrane at low operating pressure (3-15 PSI) ○ Membrane format1 enables high-throughput cellular engineering ● Integrates with existing liquid handlers ● Ready for early adopters Q1 2025 “Powered by Portal” Consumables Multiplex Delivery in Unstimulated T Cells T Cell Boost with CRISPR RNP, mRNA, and Dextran B2M expression Control Boost Mixed dextran, mRNA, & RNP Naive T cells Efficient mRNA Expression in Human iPSCs and MSCs iPSCs and MSCs boosted with GFP mRNA iPSCs Induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs) were mechanoporated with GFP mRNA. After 20h, GFP expression in iPSCs was assessed using fluorescence microscopy and flow cytometry. GFP expression in MSCs was assessed using flow cytometry and cell morphology was assessed by microscopy. Size (microns) refers to pore size used for mechanoporation. GFP expression 10 μm 11 μm GFP Transmitted light Boost Untreated Circular RNA Expression in Mixed PBMCs Day 3 cRNA GFP mRNA GFP cRNA ### -or- Mixed PBMCs were mechanoporated with either GFP mRNA or GFP cRNA (circular RNA). GFP expression was assessed on days 1, 3, 7, and 10 post-mechanoporation by flow cytometry. MSCs Mixed PBMCs Protocol T Cells Boost Gateway Activation and Expansion Edited, Activated T Cells Lovo + Clinical Scale Booster RNA expressing and edited T Cells Robust T cell expansion, viability, editing and post-boost D0 D4 D7 D10 NK boost with mRNA Mechanoporation yields efficient delivery in NK cells Unstimulated T cells were mechanoporated with β2 microglobulin (B2M) CRISPR RNPs. T cells were activated and expanded in G-Rex plates for 9 days. GFP mRNA were mechanoporated into the activated T cells at clinical scale using Portal’s MilliBooster and . Flow cytometry was conducted 48h post-boost to assess B2M knock out efficiency and efficiency of GFP expression. Robust Sequential Delivery at Clinical Scale Unstimulated T cell boost followed by activation & boost at clinical scale Unstimulated human T cells were isolated from PBMCs and boosted (mechanoporated) with a multiplexed cargo containing GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNPs. Expression of GFP mRNA and B2M protein were assessed by flow cytometry 40h post-boost. Cell Expansion Viable Cell Count (M) 5 million/well at onset Viability Viability (%) ### B2m Ko efficiency(%) Expression ### B2m Ko (%) Control Boost Control BoostD4 D7 D10 GFP expression (%) NK cells were isolated from PBMCs and mechanoporated with GFP mRNA. Viability and mRNA expression in CD3- CD56+ cells were assessed by flow cytometry 24h post-boost. NK cells ### Cd19 Car mbIL-12 Control 3 kDa Dextran Dextran delivery Expression of Ngn2 and downstream neuronal transcription factors Human iPSCs Stem-cell derived neurons iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA Boost with Ngn2 mRNA Induced pluripotent stem cells (iPSCs) were mechanoporated with neurogenin 2 (Ngn2) mRNA and dextran. Dextran delivery was assessed immediately post-boost by flow cytometry. Cells were harvested and total RNA was extracted 12h post boost. RT-qPCR was performed to assess expression levels of Ngn2 and neuronal transcription factors NeuroD1 and NeuroD4. Control Boost Control BoostControl Boost ## 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.