Poster · 2025 · ASGCT

A Next Generation Immune and Stem Cell Engineering Platform to Create Multifunctional Cells, at Clinical Scale, Using a Silicon Membrane

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

Publication text

Control Boost Control Boost Control Boost % GFP + live cells A Next Generation Immune and Stem Cell Engineering Platform to Create Multifunctional Cells, at Clinical Scale, Using a Silicon Membrane 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 Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo ● Low pressure requirements enable integration into common manufacturing equipment ● “Powered by Portal” consumables Clinical / Process Scale Integration with 3rd party cellular manufacturing Galaxy device mechanoporates cells and outputs into a trough ready for liquid handler or manual multi-channel pipette 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: ● Compatible across broad cell types including: ● Simple workflow enabling high throughput capability ● Can be used to deliver single of multiplexed cargo ● 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 Memory Survival Killing HomingmRNA siRNA ● 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 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 Research-Scale System Gateway™ Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet High Throughput Delivery Galaxy™ Beginning End 3 kDa Dextran Cell Therapy T cell Workflow which Demonstrates Sequential Cargo Delivery at Small Scale, Robust Expansion, and Large Scale Cargo Delivery using Fresenius Kabi’s Lovo® Powered by Portal with the MilliBoosterTM Unstimulated T cell boost followed by activation & boost at clinical scale Robust T Cell Expansion, Viability, Editing and mRNA Expression Post-boost Human pan-T cells were isolated from PBMCs and mechanoporated with β2 microglobulin (B2M) CRISPR RNPs using the Gateway™, R&D scale system. Post-mechanoporation cells were seeded at 5 million cells per well in a G-Rex® 6 Well Plate from ScaleReady and activated using Dynabeads. Activated T cell viability was measured using the NC-202™ Cell Counter was used to determine cell concentration and viability at 4, 7, and 10 days post-mechanoporation. Activated T cells were expanded for a total of 9 days before using the Lovo® from Fresenius Kabi with the Powered by Portal MilliBooster™attachment to deliver GFP mRNA to the cells at clinical scale. Cells were returned to culture for 48 hours immediately post-mechanportation. Flow cytometry was conducted after 48h to assess B2M knockout efficiency and GFP expression. Protocol Electroporation Mechanoporation T cells T cells Mechanical Delivery Has Limited Non-specific Impact on Normal T Cell Function q threshold Electroporation vs. Control (6hr) Mechanical Delivery vs. Control (6hr) Electroporation vs. Control (24hr) Mechanical Delivery vs. Control (24hr) 0.25 8,141 2,211 3,309 0 0.1 4,072 0 7 0 0.05 330 0 1 0 Table 1. FDR q-value thresholds for pairwise comparisons in unstimulated T cells Unstimulated human T cells were electroporation or mechanoporated. RNA was collected at 6-hours and 24 hours post-manipulation. Gene expression was evaluated by microarray and mechanoportated and electroporated samples were compared to untreated controls. Mechanical Delivery Preserves Stem Cell Function & Enables Rapid Cell Manipulation Electroporation Mechanoporation Efficient HSC Engraftment Without Rest Ctrl ElectroMech 3x CD19+ B cell Boost with transduction enhancer (TE) cRNA Circular RNA Incubate with GFP lentivirus 3 days Enabling Lentivirus Infection in CD19+ Population CD19+ cells were isolated from PBMCs and mechanoporated with a circular RNA coding for a transduction enhancer. The cells were incubated with a GFP lentivirus for 3 days post mechanoporation. Expression of the transduction enhancer and GFP were assessed using flow cytometry on day 3. VirusNo Virus CD34+ cells ● Human CD34+ cells were electroporation or mechanoporated. RNA was collected at 6-hours and 24 hours post-manipulation. Gene expression was evaluated by microarray in comparison to untreated controls. ● Human CD34 positive cells were treated by electroporation or mechanoporation and subsequently injected into mice to measure engraftment levels based on splenic chimerism. 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

Cd19 Car

mbIL-12 Control Boost Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Dextran, mRNA, &

B2m Crispr Rnp

GFP expression Unstim 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 GFP Expressing, B2M Edited, and Dextran Delivered T Cells

% Gfp+, B2m- and

Dextran+ of Live Control Boost 85.7 B2M expression GFP 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. %live (relative to UT) Untreated Human iPSCs Stem-cell derived neurons Boost with Ngn2 mRNA iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA Expression of Ngn2 and downstream neuronal transcription factors Induced pluripotent stem cells (iPSCs) were mechanoporated with neurogenin 2 (Ngn2) mRNA and dextran. 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. Mechanoporation Yields Efficient Delivery in CD14+ Monocytes CD14+ GFP-mRNA

Gfp

Boosted 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. Viability Expression of GFP-mRNA % Live %GFP+ of Live Control Boost Control Boost Robust mRNA Expression in a Variety of Immune Cells PBMCs T cells (CD3+) B cells

(cd19+)

NK cells

(cd56+)

Monocytes

(cd14+)

Gfp

Untreated GFP mRNA % Live 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. Cell Expansion Viable Cell Count (M) 5 million/well at onset Viability (%) B2M KO Efficiency mRNA Expression

B2m Ko (%)

Control Boost Control BoostD4 D7 D10 GFP expression (%)

D4 D7 D10d0

Uniform Viability and Delivery into PBMCs Using the Rotea Powered by Portal with the MilliBoosterTM PBMCs Dextran Control Beginning End Control Beginning End % Live% Dextran + of Live Delivery ● Fluorescent dextran was delivered to approximately 1 billion human peripheral blood mononuclear cells (PBMCs) using the Rotea Powered by Portal with the MilliBoosterTM in under 1 minute. ● Samples were collected at the beginning and the end of the mechanoporation process. Viability and delivery at the beginning and end of the run was measured using flow cytometry.

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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 and Stem Cell Engineering Platform to Create Multifunctional Cells, at Clinical Scale, Using a Silicon Membrane 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 and Stem Cell Engineering Platform to Create Multifunctional Cells, at Clinical Scale, Using a Silicon Membrane - Document type: Poster - Year: 2025 - Outlet: ASGCT - Cells: T cells, PBMCs, NK cells, Monocytes, B cells, iPSCs - Cargos: mRNA, CRISPR RNPs, circRNA - Page URL: https://portal.bio/publications/2025-a-next-generation-immune-and-stem-cell-engineering-platform-to-create-multifunctional--ajkF_hEAACwAc9zn - Markdown URL: https://portal.bio/publications/2025-a-next-generation-immune-and-stem-cell-engineering-platform-to-create-multifunctional--ajkF_hEAACwAc9zn.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFolpHfc4bHWipjv_PortalASGCTPoster2025.pdf ## Publication text Control Boost Control Boost Control Boost % GFP + live cells A Next Generation Immune and Stem Cell Engineering Platform to Create Multifunctional Cells, at Clinical Scale, Using a Silicon Membrane 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 Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo ● Low pressure requirements enable integration into common manufacturing equipment ● “Powered by Portal” consumables Clinical / Process Scale Integration with 3rd party cellular manufacturing Galaxy device mechanoporates cells and outputs into a trough ready for liquid handler or manual multi-channel pipette 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: ● Compatible across broad cell types including: ● Simple workflow enabling high throughput capability ● Can be used to deliver single of multiplexed cargo ● 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 Memory Survival Killing HomingmRNA siRNA ● 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 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 Research-Scale System Gateway™ Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet High Throughput Delivery Galaxy™ Beginning End 3 kDa Dextran Cell Therapy T cell Workflow which Demonstrates Sequential Cargo Delivery at Small Scale, Robust Expansion, and Large Scale Cargo Delivery using Fresenius Kabi’s Lovo® Powered by Portal with the MilliBoosterTM Unstimulated T cell boost followed by activation & boost at clinical scale Robust T Cell Expansion, Viability, Editing and mRNA Expression Post-boost Human pan-T cells were isolated from PBMCs and mechanoporated with β2 microglobulin (B2M) CRISPR RNPs using the Gateway™, R&D scale system. Post-mechanoporation cells were seeded at 5 million cells per well in a G-Rex® 6 Well Plate from ScaleReady and activated using Dynabeads. Activated T cell viability was measured using the NC-202™ Cell Counter was used to determine cell concentration and viability at 4, 7, and 10 days post-mechanoporation. Activated T cells were expanded for a total of 9 days before using the Lovo® from Fresenius Kabi with the Powered by Portal MilliBooster™attachment to deliver GFP mRNA to the cells at clinical scale. Cells were returned to culture for 48 hours immediately post-mechanportation. Flow cytometry was conducted after 48h to assess B2M knockout efficiency and GFP expression. Protocol Electroporation Mechanoporation T cells T cells Mechanical Delivery Has Limited Non-specific Impact on Normal T Cell Function q threshold Electroporation vs. Control (6hr) Mechanical Delivery vs. Control (6hr) Electroporation vs. Control (24hr) Mechanical Delivery vs. Control (24hr) 0.25 8,141 2,211 3,309 0 0.1 4,072 0 7 0 0.05 330 0 1 0 Table 1. FDR q-value thresholds for pairwise comparisons in unstimulated T cells Unstimulated human T cells were electroporation or mechanoporated. RNA was collected at 6-hours and 24 hours post-manipulation. Gene expression was evaluated by microarray and mechanoportated and electroporated samples were compared to untreated controls. Mechanical Delivery Preserves Stem Cell Function & Enables Rapid Cell Manipulation Electroporation Mechanoporation Efficient HSC Engraftment Without Rest Ctrl ElectroMech 3x CD19+ B cell Boost with transduction enhancer (TE) cRNA Circular RNA Incubate with GFP lentivirus 3 days Enabling Lentivirus Infection in CD19+ Population CD19+ cells were isolated from PBMCs and mechanoporated with a circular RNA coding for a transduction enhancer. The cells were incubated with a GFP lentivirus for 3 days post mechanoporation. Expression of the transduction enhancer and GFP were assessed using flow cytometry on day 3. VirusNo Virus CD34+ cells ● Human CD34+ cells were electroporation or mechanoporated. RNA was collected at 6-hours and 24 hours post-manipulation. Gene expression was evaluated by microarray in comparison to untreated controls. ● Human CD34 positive cells were treated by electroporation or mechanoporation and subsequently injected into mice to measure engraftment levels based on splenic chimerism. 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 ### Cd19 Car mbIL-12 Control Boost Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Dextran, mRNA, & ### B2m Crispr Rnp GFP expression Unstim 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 GFP Expressing, B2M Edited, and Dextran Delivered T Cells ### % Gfp+, B2m- and Dextran+ of Live Control Boost 85.7 B2M expression GFP 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. %live (relative to UT) Untreated Human iPSCs Stem-cell derived neurons Boost with Ngn2 mRNA iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA Expression of Ngn2 and downstream neuronal transcription factors Induced pluripotent stem cells (iPSCs) were mechanoporated with neurogenin 2 (Ngn2) mRNA and dextran. 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. Mechanoporation Yields Efficient Delivery in CD14+ Monocytes CD14+ GFP-mRNA ### Gfp Boosted 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. Viability Expression of GFP-mRNA % Live %GFP+ of Live Control Boost Control Boost Robust mRNA Expression in a Variety of Immune Cells PBMCs T cells (CD3+) B cells ### (cd19+) NK cells ### (cd56+) Monocytes ### (cd14+) ### Gfp Untreated GFP mRNA % Live 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. Cell Expansion Viable Cell Count (M) 5 million/well at onset Viability (%) B2M KO Efficiency mRNA Expression ### B2m Ko (%) Control Boost Control BoostD4 D7 D10 GFP expression (%) ### D4 D7 D10d0 Uniform Viability and Delivery into PBMCs Using the Rotea Powered by Portal with the MilliBoosterTM PBMCs Dextran Control Beginning End Control Beginning End % Live% Dextran + of Live Delivery ● Fluorescent dextran was delivered to approximately 1 billion human peripheral blood mononuclear cells (PBMCs) using the Rotea Powered by Portal with the MilliBoosterTM in under 1 minute. ● Samples were collected at the beginning and the end of the mechanoporation process. Viability and delivery at the beginning and end of the run was measured using flow cytometry. ## 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.