Poster · 2025 · AAI

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

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

Publication text

Control Boost Control Boost Control Boost ● 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 immune cell engineering platform to create multifunctional cells using a silicon membrane-based delivery system Crystal Tsui, Eleni Rogers, Zhihui Song, Sophia Hirsch, Darby Kreienberg, Andrew Larocque, Jacquelyn Hanson, Alec Barclay, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA Defined pore size for deformation Mechanical Delivery as a Universal Cell Engineering Solution Research-Scale System Gateway™ Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet 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 Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo 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. 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 Robust mRNA Expression in a Variety of Immune Cells 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 Connect with Portal! Contact us for beta program eligibility info@portal.bio

(cd3+)

B cells

(cd19+)

NK cells

(cd56+)

Monocytes

(cd14+)

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

(d6)

CD19 CAR circRNA mblL-12 circRNA

Cd19 Car

mbIL-12 Control Boost Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Mechanoporation yields efficient delivery in CD14+ monocytes CD14+ Dextran Dextran 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

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: Memory Survival Killing HomingmRNA siRNA expression Mechanoporation yields efficient delivery in NK cells GFP mRNA 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. 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 iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA Human iPSCs Stem-cell derived neurons Boost with Ngn2 mRNA 3 kDa Dextran Dextran delivery Induced pluripotent stem cells (iPSCs) were mechanoporated with neurogenin 2 (Ngn2) mRNA and dextran. Dextran delivery was assessed day0 post-boost by flow cytometry. Cells were harvested and total RNA was extracted 12h post-boost. Expression levels of Ngn2 and neuronal transcription factors NeuroD1 and NeuroD4 were assessed by RTqPCR. Expression of Ngn2 and downstream neuronal transcription factors CD14+ GFP mRNA ● Low pressure requirements enable integration into common manufacturing equipment ● “Powered by Portal” consumables Clinical / Process Scale Integration with 3rd party cellular manufacturing High Throughput Delivery Galaxy™ Galaxy device mechanoporates cells and outputs into a trough ready for liquid handler or manual multi-channel pipette Control Boost Control 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 next generation immune 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 immune cell engineering platform to create multifunctional cells using a silicon membrane-based delivery system - Document type: Poster - Year: 2025 - Outlet: AAI - Cells: T cells, iPSCs, Monocytes, NK cells, B cells, PBMCs - Cargos: mRNA, circRNA, CRISPR RNPs - Page URL: https://portal.bio/publications/2025-a-next-generation-immune-cell-engineering-platform-to-create-multifunctional-cells-us--ajkGBxEAAC4Ac90N - Markdown URL: https://portal.bio/publications/2025-a-next-generation-immune-cell-engineering-platform-to-create-multifunctional-cells-us--ajkGBxEAAC4Ac90N.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFoqGnfc4bHWipk2_AAIPoster2025.pdf ## Publication text Control Boost Control Boost Control Boost ● 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 immune cell engineering platform to create multifunctional cells using a silicon membrane-based delivery system Crystal Tsui, Eleni Rogers, Zhihui Song, Sophia Hirsch, Darby Kreienberg, Andrew Larocque, Jacquelyn Hanson, Alec Barclay, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA Defined pore size for deformation Mechanical Delivery as a Universal Cell Engineering Solution Research-Scale System Gateway™ Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet 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 Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo 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. 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 Robust mRNA Expression in a Variety of Immune Cells 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 Connect with Portal! Contact us for beta program eligibility info@portal.bio ### (cd3+) B cells ### (cd19+) NK cells ### (cd56+) Monocytes ### (cd14+) 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 ### (d6) CD19 CAR circRNA mblL-12 circRNA ### Cd19 Car mbIL-12 Control Boost Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Mechanoporation yields efficient delivery in CD14+ monocytes CD14+ Dextran Dextran 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 ### 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: Memory Survival Killing HomingmRNA siRNA expression Mechanoporation yields efficient delivery in NK cells GFP mRNA 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. 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 iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA Human iPSCs Stem-cell derived neurons Boost with Ngn2 mRNA 3 kDa Dextran Dextran delivery Induced pluripotent stem cells (iPSCs) were mechanoporated with neurogenin 2 (Ngn2) mRNA and dextran. Dextran delivery was assessed day0 post-boost by flow cytometry. Cells were harvested and total RNA was extracted 12h post-boost. Expression levels of Ngn2 and neuronal transcription factors NeuroD1 and NeuroD4 were assessed by RTqPCR. Expression of Ngn2 and downstream neuronal transcription factors CD14+ GFP mRNA ● Low pressure requirements enable integration into common manufacturing equipment ● “Powered by Portal” consumables Clinical / Process Scale Integration with 3rd party cellular manufacturing High Throughput Delivery Galaxy™ Galaxy device mechanoporates cells and outputs into a trough ready for liquid handler or manual multi-channel pipette Control Boost Control 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.