# A platform for multiplexed RNA-based and genomic cell engineering for cancer immunotherapy 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 platform for multiplexed RNA-based and genomic cell engineering for cancer immunotherapy - Document type: Poster - Year: 2024 - Outlet: SITC 2024 - Cells: T cells, PBMCs, Monocytes, iPSCs - Cargos: mRNA, CRISPR RNPs, circRNA - Page URL: https://portal.bio/publications/2024-a-platform-for-multiplexed-rna-based-and-genomic-cell-engineering-for-cancer-immunoth--ajkGRBEAACoAc93C - Markdown URL: https://portal.bio/publications/2024-a-platform-for-multiplexed-rna-based-and-genomic-cell-engineering-for-cancer-immunoth--ajkGRBEAACoAc93C.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFotOnfc4bHWiplS_SITC2024Poster.pdf ## Publication text ● Portal’s mechanoporation technology can be used for RNA-based cell engineering ○ Compatible across broad cell types including: ○ Efficient delivery of many cargo types including: ### Summary Research-Scale System Gateway™ High-Throughput Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet Powered by Portal™ Consumables Introduction to Portal’s Approach: Implementing a Universal Cell Engineering Solution ● Portal is advancing a transformative technology for cellular engineering based on mechanical deformation that allows cytosolic delivery of a multitude of cargoes including mRNA, CRISPR RNPs, peptides, DELs and others1-4 ● The technology is compatible with many primary cell types including immune and stem cells as well as cell lines ● Cells are engineered (‘boosted’) through a membrane at low operating pressure (3-15 PSI) ● Membrane format5 enables high-throughput cellular engineering A platform for multiplexed RNA-based and genomic cell engineering for cancer immunotherapy Eleni Rogers, Zhihui Song, Sophia Hirsch, Rachel Conover, Andrew Larocque, Darby Kreienberg, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA (1) Sharei et al., PNAS, 2013 (2) Szeto et al., Scientific Reports, 2015 (3) Sharei et al., PLOS One, 2015 (4) Sharei et al., Integrative Biology, 2014 (5) Williams AR, Bao S, Miller DL, Biotechnol Bioeng., 1999 Connect with Portal! Contact us for beta program eligibility info@portal.bio ● Low pressure requirements enable integration into common manufacturing equipment ● Powered by Portal™ consumables Clinical / Process Scale Integration with 3rd party cellular manufacturing Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo ● Integrates with existing liquid handlers ● Ready for early adopters late 2024 Mechanoporation yields efficient delivery in NK cells Robust Sequential Delivery at Clinical Scale Unstimulated T cell boost followed by activation & boost at clinical scale Cell Expansion Viable Cell Count (M) 5 million/well at onset Viability Viability (%) B2M KO efficiency(%) mRNA Expression ### B2m Ko (%) Control Boost Control BoostD4 D7 D10 GFP expression (%) Robust T cell expansion, viability, editing and mRNA expression post-boost Circular RNA Expression in Mixed PBMCs Mixed PBMCs GFP mRNA GFP cRNA ### -or- NK boost with mRNA 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. Day 3 mRNA expression cRNA Mixed PBMCs were mechanoporated with either GFP mRNA or GFP cRNA (circular RNA). GFP expression was assessed on days 1, 3, 7, and 10 post-mechanoporation by flow cytometry. Unstimulated T cells were mechanoporated with β2 microglobulin (B2M) CRISPR RNPs. T cells were activated and expanded in G-Rex plates for 9 days. GFP mRNA were mechanoporated into the activated T cells at clinical scale using Portal’s MilliBooster. Flow cytometry was conducted 48h post-boost to assess B2M knockout efficiency and efficiency of GFP expression. Activated T cells Engineered with Simultaneous circRNA Delivery for IL-12 and CD19 CAR Activated T cells ### (d6) CD19 CAR circRNA mblL-12 circRNA CD19 CAR mbIL-12 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. Peripheral blood mononucleated cell (PBMC) boost with mRNA Efficient Delivery of mRNA to a variety of immune cell types 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 T cells ### (cd3+) B cells ### (cd19+) NK cells ### (cd56+) Monocytes ### (cd14+) PBMCs GFP mRNA Protocol Multiplex Delivery in Unstimulated T Cells T Cell Boost with CRISPR RNP, mRNA, and Dextran Mixed dextran, mRNA, & RNP Naive T cells B2M expression GFP expression Unstimulated human T cells were isolated from PBMCs and boosted (mechanoporated) with a multiplexed cargo containing GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNPs. Expression of GFP mRNA and B2M protein were assessed by flow cytometry 40h post-boost. iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA Mechanoporation yields efficient delivery in CD14+ monocytes 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. Dextran ### Cd14+ Dextran GFP mRNA ### -or- NK cells HSCs B cellsPBMCs T cells iPSCs ● Portal’s Research Scale Gateway™ enables complex cell engineering with a simple workflow ● Little impact on normal cellular function ● Portal plans to use its technology for transient modifications of cells for: ○ Cell therapies ○ Biomanufacturing ○ Regenerative medicine ○ Diagnostics and Analysis ○ High throughput screening ### Crispr RNPs mRNA, circRNA, siRNA Antibodies, proteins, peptides Memory Survival Killing HomingmRNA siRNA Future Directions: Using Portal’s Mechanoporation Technology for Transient Enhancement of Cell Function Boost immune cells to improve effector function for cell therapy mRNA encoded membrane bound (mb) IL-2/12 cytokines were boosted into T cells and implanted into mice. Blood was drawn after 5 days and % of T cells expressing mbIL-2/12 was measured, illustrating potential to improve memory phenotype and cytotoxicity in a mouse model. TCM-like TEM-like TEFF-like Silver et al, ESMO 2022 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 ## 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.