Poster · 2025 · ISCT: International Society for Cell & Gene Therapy

A next generation cell engineering platform to create multifunctional cells using a silicon membrane-based delivery system

Cells: T cells, PBMCs, Monocytes, NK cells
Cargos: mRNA, CRISPR RNPs, circRNA

Publication text

● Portal is advancing a transformative technology for cellular engineering compatible across cell types and cargos ○ mRNA, CRISPR RNPs, peptides, proteins, siRNA, and many others1-4 ○ Primary and patient-derived 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 A next generation cell engineering platform to create multifunctional cells using a silicon membrane-based delivery system Zhihui Song, Sophia Hirsch, Eleni Rogers, Darby Kreienberg, Andrew Larocque, Jacquelyn Hanson, Anil Narasimha, Alec Barclay, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA Research-Scale System Gateway™ Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet Defined pore size for deformation Mechanical Delivery as a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo 1Sharei, et al., PNAS, 2013; 2Szeto, et al., Scientific Reports, 2015; 3Sharei, et al., PLOS One, 2015; 4Sharei et al., Integrative Biology 2014; 5Williams AR, Bao S, Miller DL., Biotechnol Bioeng. 1999 ● Low pressure requirements enable integration into common manufacturing equipment ● “Powered by Portal” consumables Clinical / Process Scale Integration with 3rd party cellular manufacturing PBMCs 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. Robust mRNA Expression in a Variety of Immune Cells Connect with Portal! Contact us for beta program eligibility info@portal.bio

Summary

● Portal’s mechanoporation technology can be used for drug discovery and cell engineering ● Efficient delivery of diverse impermeable molecules for use in downstream screening including: ● Simple workflow enabling high throughput capabilities ● Can be used to deliver single or multiplexed cargoes ● Little impact on normal cellular function or morphology CRISPR RNPAntibodies mRNA, circRNA, siRNA NK cells HSCs B cellsPBMCs T cells iPSCs HeLa cells HEK293 cells + Other cell lines ● Compatible across broad cell types including:

(cd3+)

B cells

(cd19+)

NK cells

(cd56+)

Monocytes

(cd14+)

Gfp

High Throughput Delivery Galaxy™ Galaxy device mechanoporates cells and outputs into a trough ready for liquid handler or manual multi-channel pipette Memory Survival Killing HomingmRNA siRNA Mechanoporation yields efficient delivery in CD14+ monocytes CD14+ Dextran GFP mRNA

-or-

Dextran

Gfp

CD14+ monocytes isolated from a leukopak were mechanoporated with dextran or GFP-mRNA. Viability and dextran were measured on day 0 by flow cytometry. Viability and GFP expression was measured on day 1 by flow cytometry. Expression of GFP-mRNA in CD14+Viability of CD14+ Viability of CD14+ Delivery of Dextran to CD14+ % live cells% live cells % GFP+ of live cells % Dextran+ of live Boosted Boosted Mechanoporation yields efficient delivery in NK cells GFP mRNA 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. Unstimulated T cells were mechanoporated with β2 microglobulin (B2M) CRISPR RNP using the research scale MicroBooster. T cells were activated and expanded in G-Rex plates for 9 days. Activated T cells were mechanoporated with GFP mRNA at clinical scale using the Lovo as the driving force. Flow cytometry was conducted 48h post-boost to assess B2M protein expression and efficiency of GFP expression. Clinical Scale Workflow Featuring Sequential Mechanoporation, Robust Editing, mRNA Expression and T Cell Expansion T Cells Boost Gateway Activation and Expansion Edited, Activated T Cells Lovo + Clinical Scale Booster RNA expressing and edited T Cells B2M Knock Out efficiency(%) B2M Knock Out (%) mRNA Expression Cell Expansion Viable Cell Count (M) 5 million/well at onset

D0 D4 D7 D10

Viability Viability (%)

D4 D7 D10

% GFP + live cells CD19+ B cell Boost with transduction enhancer (TE) cRNA cRNA Incubate with GFP lentivirus 3 days Enabling Lentivirus infection on CD19+ population CD19+ cells were isolated from PBMCs and mechanoporated with a circular RNA coding for a transduction enhancer (TE). The boosted cells were then incubated with a GFP lentivirus for 3 days. Surface receptor and GFP expression were assessed using flow cytometry after incubation period. VirusNo Virus % GFP + live cells Activation and expansion

Trac & B2m

CRISPR RNPs Multiplexed CRISPR Knock Out in Unstimulated T cells TRAC expression B2M expression Unstimulated T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing CRISPR RNPs targeting T Cell Receptor Alpha Constant (TRAC) and β2 microglobulin (B2M). T cells were activated and expanded for 4 days and expression of TRAC and B2M protein were assessed by flow cytometry. Viability of Activated T Cells Editing Efficiency Dextran, mRNA, &

B2m Crispr Rnp

GFP expression B2M expression 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. Robust Multiplex Delivery in Unstimulated T Cells T cells Engineered with Simultaneous circRNA Delivery for IL-12, IL-2 and CD19 CAR Activated

(d6)

mbIL-2 circRNA mblL-12 circRNA CD19 CAR circRNA mbIL-2 mbIL-12

Cd62l

Cd127

Cd19 Car

Activated T cells were mechanoporated with membrane bound IL-2 (mbIL-2) ,mbIL-12 and CD19 CAR cRNA. Flow cytometry was used to assess CAR, IL2 and IL12 expression 24h postboost; CD127 and CD62L expression 96 h post boost. Phase contrast images show proliferation of T cells 24 hours post-boost. Scale bar (white) = 300 μm. To assess cytotoxicity, boosted activated T cells (left) and unstimulated T cells (right) were rested for 2 h before co-culturing with Raji cells at different ratios of effector cells (E) to target cells (T). Cytotoxicity was measured 48h post-boost by quantifying reduction in CD19+ Raji cells relative to counting beads.

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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 next generation cell engineering platform to create multifunctional cells using a silicon membrane-based delivery system 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 next generation cell engineering platform to create multifunctional cells using a silicon membrane-based delivery system - Document type: Poster - Year: 2025 - Outlet: ISCT: International Society for Cell & Gene Therapy - Cells: T cells, PBMCs, Monocytes, NK cells - Cargos: mRNA, CRISPR RNPs, circRNA - Page URL: https://portal.bio/publications/2025-a-next-generation-cell-engineering-platform-to-create-multifunctional-cells-using-a-s--ajkGAhEAACwAc9z- - Markdown URL: https://portal.bio/publications/2025-a-next-generation-cell-engineering-platform-to-create-multifunctional-cells-using-a-s--ajkGAhEAACwAc9z-.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFop23fc4bHWipky_ISCT_2025_Poster.pdf ## Publication text ● Portal is advancing a transformative technology for cellular engineering compatible across cell types and cargos ○ mRNA, CRISPR RNPs, peptides, proteins, siRNA, and many others1-4 ○ Primary and patient-derived 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 A next generation cell engineering platform to create multifunctional cells using a silicon membrane-based delivery system Zhihui Song, Sophia Hirsch, Eleni Rogers, Darby Kreienberg, Andrew Larocque, Jacquelyn Hanson, Anil Narasimha, Alec Barclay, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA Research-Scale System Gateway™ Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet Defined pore size for deformation Mechanical Delivery as a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo 1Sharei, et al., PNAS, 2013; 2Szeto, et al., Scientific Reports, 2015; 3Sharei, et al., PLOS One, 2015; 4Sharei et al., Integrative Biology 2014; 5Williams AR, Bao S, Miller DL., Biotechnol Bioeng. 1999 ● Low pressure requirements enable integration into common manufacturing equipment ● “Powered by Portal” consumables Clinical / Process Scale Integration with 3rd party cellular manufacturing PBMCs 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. Robust mRNA Expression in a Variety of Immune Cells Connect with Portal! Contact us for beta program eligibility info@portal.bio ### Summary ● Portal’s mechanoporation technology can be used for drug discovery and cell engineering ● Efficient delivery of diverse impermeable molecules for use in downstream screening including: ● Simple workflow enabling high throughput capabilities ● Can be used to deliver single or multiplexed cargoes ● Little impact on normal cellular function or morphology CRISPR RNPAntibodies mRNA, circRNA, siRNA NK cells HSCs B cellsPBMCs T cells iPSCs HeLa cells HEK293 cells + Other cell lines ● Compatible across broad cell types including: ### (cd3+) B cells ### (cd19+) NK cells ### (cd56+) Monocytes ### (cd14+) ### Gfp High Throughput Delivery Galaxy™ Galaxy device mechanoporates cells and outputs into a trough ready for liquid handler or manual multi-channel pipette Memory Survival Killing HomingmRNA siRNA Mechanoporation yields efficient delivery in CD14+ monocytes CD14+ Dextran GFP mRNA ### -or- Dextran ### Gfp CD14+ monocytes isolated from a leukopak were mechanoporated with dextran or GFP-mRNA. Viability and dextran were measured on day 0 by flow cytometry. Viability and GFP expression was measured on day 1 by flow cytometry. Expression of GFP-mRNA in CD14+Viability of CD14+ Viability of CD14+ Delivery of Dextran to CD14+ % live cells% live cells % GFP+ of live cells % Dextran+ of live Boosted Boosted Mechanoporation yields efficient delivery in NK cells GFP mRNA 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. Unstimulated T cells were mechanoporated with β2 microglobulin (B2M) CRISPR RNP using the research scale MicroBooster. T cells were activated and expanded in G-Rex plates for 9 days. Activated T cells were mechanoporated with GFP mRNA at clinical scale using the Lovo as the driving force. Flow cytometry was conducted 48h post-boost to assess B2M protein expression and efficiency of GFP expression. Clinical Scale Workflow Featuring Sequential Mechanoporation, Robust Editing, mRNA Expression and T Cell Expansion T Cells Boost Gateway Activation and Expansion Edited, Activated T Cells Lovo + Clinical Scale Booster RNA expressing and edited T Cells B2M Knock Out efficiency(%) B2M Knock Out (%) mRNA Expression Cell Expansion Viable Cell Count (M) 5 million/well at onset ### D0 D4 D7 D10 Viability Viability (%) ### D4 D7 D10 % GFP + live cells CD19+ B cell Boost with transduction enhancer (TE) cRNA cRNA Incubate with GFP lentivirus 3 days Enabling Lentivirus infection on CD19+ population CD19+ cells were isolated from PBMCs and mechanoporated with a circular RNA coding for a transduction enhancer (TE). The boosted cells were then incubated with a GFP lentivirus for 3 days. Surface receptor and GFP expression were assessed using flow cytometry after incubation period. VirusNo Virus % GFP + live cells Activation and expansion ### Trac & B2m CRISPR RNPs Multiplexed CRISPR Knock Out in Unstimulated T cells TRAC expression B2M expression Unstimulated T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing CRISPR RNPs targeting T Cell Receptor Alpha Constant (TRAC) and β2 microglobulin (B2M). T cells were activated and expanded for 4 days and expression of TRAC and B2M protein were assessed by flow cytometry. Viability of Activated T Cells Editing Efficiency Dextran, mRNA, & ### B2m Crispr Rnp GFP expression B2M expression 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. Robust Multiplex Delivery in Unstimulated T Cells T cells Engineered with Simultaneous circRNA Delivery for IL-12, IL-2 and CD19 CAR Activated ### (d6) mbIL-2 circRNA mblL-12 circRNA CD19 CAR circRNA mbIL-2 mbIL-12 ### Cd62l ### Cd127 ### Cd19 Car Activated T cells were mechanoporated with membrane bound IL-2 (mbIL-2) ,mbIL-12 and CD19 CAR cRNA. Flow cytometry was used to assess CAR, IL2 and IL12 expression 24h postboost; CD127 and CD62L expression 96 h post boost. Phase contrast images show proliferation of T cells 24 hours post-boost. Scale bar (white) = 300 μm. To assess cytotoxicity, boosted activated T cells (left) and unstimulated T cells (right) were rested for 2 h before co-culturing with Raji cells at different ratios of effector cells (E) to target cells (T). Cytotoxicity was measured 48h post-boost by quantifying reduction in CD19+ Raji cells relative to counting beads. ## 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.