# A next generation immune cell engineering platform to generate 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 generate multifunctional cells using a silicon membrane-based delivery system - Document type: Poster - Year: 2025 - Outlet: Keystone Symposia: Cancer Immunotherapy/Immune Cell Therapies - Cells: PBMCs, B cells, T cells, NK cells, Monocytes - Cargos: mRNA, CRISPR RNPs, circRNA - Page URL: https://portal.bio/publications/2025-a-next-generation-immune-cell-engineering-platform-to-generate-multifunctional-cells--ajkGIBEAACoAc91L - Markdown URL: https://portal.bio/publications/2025-a-next-generation-immune-cell-engineering-platform-to-generate-multifunctional-cells--ajkGIBEAACoAc91L.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFoq53fc4bHWiplA_PortalKeystoneCancerImmunoTherapyPoster.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 immune cell engineering platform to generate multifunctional cells using a silicon membrane-based delivery system 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 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. 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. 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 Robust Multiplex Delivery in Unstimulated T Cells CD19+ B cell Boost with transduction enhancer (TE) cRNA cRNA Incubate with GFP lentivirus 3 days Enabling Lentivirus infection on CD19+ population Mechanoporation yields efficient delivery in NK 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: 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. ### (cd3+) B cells ### (cd19+) NK cells ### (cd56+) Monocytes ### (cd14+) High Throughput Delivery VirusNo Virus Galaxy™ Galaxy device mechanoporates cells and outputs into a trough ready for liquid handler or manual multi-channel pipette 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. 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 Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR 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 % GFP + live cells % GFP + live cells ## 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.