# Multiplexed genomic and RNA cell engineering in human stem and immune cells via mechanoporation 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: Multiplexed genomic and RNA cell engineering in human stem and immune cells via mechanoporation - Document type: Poster - Year: 2025 - Outlet: Keystone Symposia: Precision Genome Engineering - Cells: T cells, B cells, iPSCs, PBMCs - Cargos: circRNA, CRISPR RNPs, mRNA - Page URL: https://portal.bio/publications/2025-multiplexed-genomic-and-rna-cell-engineering-in-human-stem-and-immune-cells-via-mecha--ajkGLREAACgAc912 - Markdown URL: https://portal.bio/publications/2025-multiplexed-genomic-and-rna-cell-engineering-in-human-stem-and-immune-cells-via-mecha--ajkGLREAACgAc912.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFor0Hfc4bHWiplF_PortalKeystonePGEPoster.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 Multiplexed genomic and RNA cell engineering in human stem and immune cells via mechanoporation Darby Kreienberg, Eleni Rogers, Zhihui Song, Sophia Hirsch, 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 KO efficiency(%) ### B2m Ko (%) Control Boost mRNA Expression Control Boost 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 Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Human Pan-T cells were isolated and activated for 6 days. Activated T cells were mechanoporated with CD19 CAR circRNA and membrane bound IL-12 (mbIL-12) circRNA. Boosted cells were co-cultured 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). Activated ### (d6) CD19 CAR circRNA mblL-12 circRNA ### Cd19 Car mbIL-12 Control CD19+ B cell Boost with surface receptor cRNA cRNA Incubate with GFP lentivirus 3 days Enabling Lentivirus infection on CD19+ population Efficient mRNA Expression in Human iPSCs iPSCs boosted with GFP mRNA GFP mRNA iPSCs Induced pluripotent stem cells (iPSCs) were mechanoporated with GFP mRNA. After 20h, GFP expression in iPSCs was assessed using fluorescence microscopy and flow cytometry. GFP Transmitted light Boost Untreated ### 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 DELs Peptides Macrocycle Antibodies Small molecules NK cells HSCs B cellsPBMCs T cells iPSCs HeLa cells HEK293 cells + Other cell lines Connect with Portal! Contact us for beta program eligibility info@portal.bio ● Compatible across broad cell types including: CD19+ cells were isolated from PBMCs and mechanoporated with a circular RNA coding for a surface receptor. 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. 3 kDa Dextran Dextran delivery Expression of Ngn2 and downstream neuronal transcription factors Human iPSCs Stem-cell derived neurons iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA Boost with Ngn2 mRNA Induced pluripotent stem cells (iPSCs) were mechanoporated with neurogenin 2 (Ngn2) mRNA and dextran. Dextran delivery was assessed immediately post-boost by flow cytometry. Cells were harvested and total RNA was extracted 12h post boost. RT-qPCR was performed to assess expression levels of Ngn2 and neuronal transcription factors NeuroD1 and NeuroD4. Control Boost ControlControl Boost ### (cd3+) B cells ### (cd19+) NK cells ### (cd56+) Monocytes ### (cd14+) ### Gfp Expression of Ngn2 and downstream neuronal transcription factors High Throughput Delivery No Virus Virus VirusNo Virus Galaxy™ Galaxy device mechanoporates cells and outputs into a trough ready for liquid handler or manual multi-channel pipette ## 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.