Publication

# Publication 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: Publication - Document type: Poster - Year: 2024 - Outlet: Tumor immunology-Immunotherapy/AACR - Cells: T cells, Monocytes, NK cells, iPSCs, PBMCs, CD34+ - Cargos: mRNA, CRISPR RNPs, siRNA, circRNA - Page URL: https://portal.bio/publications/2024-publication--ajkGTREAAC0Ac93e - Markdown URL: https://portal.bio/publications/2024-publication--ajkGTREAAC0Ac93e.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFotv3fc4bHWiplY_AACRTumorimmunology2024.pdf ## Summary Enhanced immune cell engineering utilizing a novel, effective intracellular delivery method for introduction of diverse cargo types ## Publication text Enhanced immune cell engineering utilizing a novel, effective intracellular delivery method for introduction of diverse cargo types Zhihui Song, Eleni Rogers, Sophia Hirsch, Andrew Larocque, Darby Kreienberg, Rachel Conover, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA Robust Multiplex Delivery in Unstimulated T Cells Untreated Dextran, mRNA, & ### B2m Crispr Rnp 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. Connect with Portal!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 ### Gfp Bcl2 ### G101v ### Today ### 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 High Knock Down Efficiency in CD34+ Cells siRNA CD34+ cells siRNA targeting β2 microglobulin (B2M) was mechanoporated into human CD34+ hematopoietic stem cells. Flow cytometry was conducted after 40 hours to assess viability and B2M expression. Research-Scale System Gateway™ Consumable Delivery Cartridge Compact bench-top unit that controls delivery parameters and easily fits in biosafety cabinet MicroBooster™ Defined pore size for deformation Cell type-specific consumable for optimal mechanoporationmediated cargo delivery 18mm 35mm Introduction to Portal’s Approach: Implementing a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo ● 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 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 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. TCM-like TEM-like TEFF-like Boost immune cells to improve effector function for cell therapy Future Directions: Using Portal’s Mechanoporation Technology for Transient Enhancement of Cell Function 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 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. Efficient delivery of mRNA to a variety of immune cell types ### Summary ● 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: ● Portal’s Research Scale Gateway™ enables complex cell engineering with a simple workflow ○ Can be used to deliver multiplexed cargo ● Little impact on normal cellular function or morphology ● Portal plans to use its technology for transient modifications of cells for: ○ Cell therapies ○ Biomanufacturing ○ Regenerative medicine ○ Diagnostics and Analysis ○ High throughput screening Memory Survival Killing HomingmRNA siRNA Pan-T cells were activated with Dynabeads which were removed after 48 hours. On Day 6 of activation, the T cells were mechanoporated with circRNA encoding CD19 CAR and either mbIL-2 or mbIL-12. After a 2-hour rest, the boosted cells were co-cultured with Raji cells at varying ratios. Following 2 days of co-culture, Raji cells were counted using counting beads to determine killing efficiency. Activated T cells ### (d6) ### Cd19 Car cRNA + mbIL-2 cRNA OR mbIL-12 cRNA Efficient mRNA Expression in Human iPSCs GFP mRNA iPSCs Induced pluripotent stem cells (iPSCs) were mechanoporated with GFP mRNA. After 20h, GFP expression was assessed using fluorescence microscopy and flow cytometry. GFP Transmitted light Boost Untreated 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 Control Boost Control 3 kDa Dextran Dextran delivery Ngn2 Human stem cells (iPSCs) Human stem-cell derived neurons iPSCs express early neuronal markers 12hrs after boosting with Ngn2 mRNA T Cells Boost Gateway Activation and Expansion Edited, Activated T Cells Lovo + Clinical Scale BoosterRNA expressing and edited T Cells B2M KO efficiency(%) mRNA Expression ### B2m Ko (%) Control Boost Control Boost Cell Expansion Viable Cell Count (M) 5 million/well at onset ### D0 D4 D7 D10 Viability Viability (%) ### D4 D7 D10 ### Gfp Dextran Mechanoporation yields efficient delivery in CD14+ monocytes Dextran OR mRNA Efficient delivery of GFP mRNA to NK cells NK cells GFP mRNA was mechanoporated into natural killer cells (NKs). Viability was assessed by flow cytometry on the same day, and GFP expression was assessed by flow cytometry the following day after boost. Activation and expansion T cells ### Trac & B2m CRISPR RNPs Multiplexed CRISPR KO in unstimulated T cells TRAC 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 protein, B2M protein were assessed by flow cytometry. Viability of Activated T Cells Editing Efficiency Dextran or GFP mRNA were mechanoporated into isolated CD14+ monocytes isolated from a leukopak. Flow cytometry was used to assess Viability and dextran delivery on the same day as mechanoporation, and to assess mRNA expression along with viability the next day following mechanoporation. NK cells HSCs MonocytesPBMCs T cells iPSCs HeLa ### Hek Activated T cells engineered with circRNA for CD19 CAR + membrane bound IL-2 or IL-12 ### Cd19 Car ### Il-12 Control Human iPSCs were boosted with Ngn2 mRNA. Cells were harvested at 12 hours and Ngn2, NeuroD1, & NeuroD4 expression were assessed via RT-qPCR. ### Crispr RNPs mRNA, circRNA, siRNA Antibodies, proteins, peptides GFP mRNA mbIL-2 ### Car ## 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.