Poster · 2025 · AACR

Combining circular RNA and a silicon membrane-based cell engineering approach to create CAR-Ts with membrane bound IL-2 and IL-12

Cells: T cells, PBMCs, Monocytes, NK cells, B 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 Combining circular RNA and a silicon membrane-based cell engineering approach to create CAR-Ts with membrane bound IL-2 and IL-12 Zhihui Song, Andrew Larocque, Sophia Hirsch, Eleni Rogers, Darby Kreienberg, Jacquelyn Hanson, 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+)

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 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 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.

# Combining circular RNA and a silicon membrane-based cell engineering approach to create CAR-Ts with membrane bound IL-2 and IL-12 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: Combining circular RNA and a silicon membrane-based cell engineering approach to create CAR-Ts with membrane bound IL-2 and IL-12 - Document type: Poster - Year: 2025 - Outlet: AACR - Cells: T cells, PBMCs, Monocytes, NK cells, B cells - Cargos: mRNA, CRISPR RNPs, circRNA - Page URL: https://portal.bio/publications/2025-combining-circular-rna-and-a-silicon-membrane-based-cell-engineering-approach-to-crea--ajkGDBEAACwAc90Z - Markdown URL: https://portal.bio/publications/2025-combining-circular-rna-and-a-silicon-membrane-based-cell-engineering-approach-to-crea--ajkGDBEAACwAc90Z.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFoqS3fc4bHWipk5_AACR_ImmunoTherapy_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 Combining circular RNA and a silicon membrane-based cell engineering approach to create CAR-Ts with membrane bound IL-2 and IL-12 Zhihui Song, Andrew Larocque, Sophia Hirsch, Eleni Rogers, Darby Kreienberg, Jacquelyn Hanson, 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+) 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 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 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.