# A platform for RNA-based and genomic cell engineering: simplifying multiplexed delivery to human primary immune and stem cells 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 RNA-based and genomic cell engineering: simplifying multiplexed delivery to human primary immune and stem cells - Document type: Poster - Year: 2024 - Outlet: ASGCT: American Society of Gene and Cell Therapy - Cells: T cells, iPSCs, PBMCs, CD34+ - Cargos: mRNA, CRISPR RNPs, siRNA - Page URL: https://portal.bio/publications/2024-a-platform-for-rna-based-and-genomic-cell-engineering-simplifying-multiplexed-deliver--ajkGYBEAACoAc94Y - Markdown URL: https://portal.bio/publications/2024-a-platform-for-rna-based-and-genomic-cell-engineering-simplifying-multiplexed-deliver--ajkGYBEAACoAc94Y.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFouR3fc4bHWiplc_KeystonePosterEmergingCellularTherapies2024-1-.pdf ## Publication text PBMCs Robust mRNA expression in a variety of immune cells GFP mRNA Boost T cells ### (cd3+) B cells ### (cd19+) NK cells ### (cd56+) Monocytes ### (cd14+) ### Gfp Peripheral blood mononucleated cell (PBMC) boost with mRNA Robust Multiplex Delivery in Unstimulated T Cells T cell boost with CRISPR RNP, mRNA, and Dextran Control GFP expression B2M expression Boost Mixed dextran, mRNA, & RNP Naive T cells High Knock Down Efficiency in CD34+ Cells Hematopoietic Stem Cell (HSC) boost with siRNA siRNA CD34+ cells Efficient mRNA Expression in Human iPSCs Induced pluripotent stem cell (iPSC) boost with GFP mRNA GFP mRNA B2M expression Boost 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. Naive T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNPs. After 40h, expression of mRNA and B2M protein were assessed by flow cytometry. β2 microglobulin (B2M) siRNA was mechanoporated into human CD34+ hematopoietic stem cells. Flow cytometry was conducted after 40 hours to assess viability and B2M expression. iPSCs Induced pluripotent stem cells (iPSCs) were lifted into single cell suspension and mechanoporated with GFP mRNA. After 20h, GFP expression was assessed using fluorescence microscopy and flow cytometry. Future directions: Using Portal’s Mechanoporation Technology for Eliminating Preconditioning Mutant BCL2 enables resistance to Venetoclax-induced apoptosis and promotes HSC survival No Treatment Venetoclax ### Gfp Bcl2 ### G101v Transferred cell frequency (% of human cells) Boost HSCs to increase homing and cell survival towards reducing pre-conditioning for cell therapies CD34+ cells were mechanoporated with either GFP or BCL-2 (G101V) mRNA and transferred to NSG mice with or without Venetoclax treatment. Bone marrow was harvested and frequency of transplanted HSCs was measured. Silva et al, ASH 2022 Silver et al, ESMO 2022 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. TIL Phenotype (Blood, Day 5) TCM-like TEM-like TEFF-like Boost immune cells to improve effector function for cell therapy ### Summary ● Portal’s mechanoporation technology can be used for RNA-based cell engineering ○ Compatible across broad cell types including: ○ Can be used to deliver single RNAs or multiplexed cargoes ● 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 NK cells HSCs B cellsPBMCs T cells iPSCs GFP Transmitted light Boost Control Research-Scale System Gateway™ Consumable Delivery Cartridge Compact bench-top unit that controls delivery parameters and easily fits in hood MicroBooster™ Defined pore size for deformation 18mm 35mm Cell type-specific consumable for optimal mechanoporationmediated cargo delivery Introduction to Portal’s Approach: Implementing A Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular Delivery Memory Survival Killing Homing mRNA siRNA Cells and Cargo Cells Cargo 1. Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999 ● Low pressure requirements enable integration into common manufacturing equipment ● “Powered by Portal” consumables Clinical / Process Scale Integrate with 3rd Party cellular manufacturing ● Portal is creating a transformative technology for cellular engineering compatible across cell types and cargo types ○ Primary and patient-derived cell lines including immune and stem cells ○ mRNA, siRNA, saRNA, DNA, RNPs, proteins, and other cargos ● Cells are boosted through a membrane at low operating pressure (3-15 PSI) ○ Membrane format1 enables high-throughput cellular engineering ### Gfp Bcl2 ### G101v A Platform for RNA-based Cell Engineering: Simplifying Multiplexed Delivery to Human Primary Immune and Stem Cells Sophia Hirsch, Zhihui Song, Rachel Conover, Lina Abu-Absi, Athira Pillai, Andrew Larocque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA Induced pluripotent stem cells (iPSCs) were mechanoporated with Ascl1 saRNA and mRNA for 5 neuronal transcription factors (FoxA2, Lmx1a, Nurr1, Pitx3, EN1). 14d post mechanoporation, cells were positive for dopaminergic neuronal marker tyrosine hydroxylase and mature neuronal marker MAP2 (green arrows) . Future directions: Using Portal’s Mechanoporation Technology to Drive Cellular Reprogramming Boost iPSCs to drive neuronal differentiation Wu et al, ISSCR 2022 CD19 chimeric antigen receptor (CAR) mRNA was mechanoporated into activated T cells. 20h post-boost, flow cytometry was conducted to assess CD19 CAR expression. Boosted T cells were mixed with Raji cells and a Cytotox96 assay was conducted to measure LDH release to assess CAR effector function. Activated T cells ### Cd19-car mRNA CD4 expression CAR expression ### Cd19-car Effector : Target 27:1 CD19-CAR Activity by mRNA Delivery in Activated T Cells Efficient Delivery of mRNA to a variety of immune cell types 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 Background on Mechanoporation ● Previous work has shown that mechanical deformation of cells through a microfluidic channel enables cytosolic intracellular delivery1 ○ Delivers macromolecules2,3 to immune and other primary cell types ● Cells and cargo are mixed together then squeezed through a narrow channel at high pressure. The cell membrane is temporarily disrupted, allowing the target cargo to enter the cytosol before the membrane seals4. Connect with Portal! Future directions: Using Portal’s Mechanoporation Technology for Transient Enhancement of Cell Function ### Toda ### Y Future By engineering the cell, not the patient we can increase safety, reduce costs, and expand access Toxic chemotherapy pre-conditioning can lead to: - Death, infertility, and risk of infection - Often a week or more in hospitalization Cells engraft with no or mild preconditioning Toxicity, side effects, hospitalizations Cells engraft in immune depleted patient ### Engraftmentpre-conditioning Contact us for beta program eligibility info@portal.bio Activated T cell boost with CD19-CAR mRNA ## 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.