# A Drug Discovery Platform Enabling Robust Intracellular Delivery of Cell Impermeable Cargo 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: A Drug Discovery Platform Enabling Robust Intracellular Delivery of Cell Impermeable Cargo via Mechanoporation - Document type: Poster - Year: 2024 - Outlet: Drug Discovery Chemistry - Cells: T cells, iPSCs, Cancer Cell Lines - Cargos: mRNA, CRISPR RNPs, DELs, Antibody, Oligo, Macrocycle, Peptide, small molecules - Page URL: https://portal.bio/publications/2024-a-drug-discovery-platform-enabling-robust-intracellular-delivery-of-cell-impermeable--ajkGaBEAACwAc95E - Markdown URL: https://portal.bio/publications/2024-a-drug-discovery-platform-enabling-robust-intracellular-delivery-of-cell-impermeable--ajkGaBEAACwAc95E.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFmhk3fc4bHWioZh_Hirsch_DrugDiscoveryChemistryPoster.pdf ## Summary Sophia Hirsch, Zhihui Song, Andrew Larocque, Rachel Conover, Athira Pillai, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei ## Publication text 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 DeliveryCells and Cargo Cells Cargo ● Low pressure requirements enable integration into existing robotics ● Ready for early adopters High-Throughput Capabilities “Powered by Portal” Consumables ● Portal is creating a transformative technology for cellular engineering compatible across cell types and cargos ○ Small molecules, oligos, peptides, antibodies, DELs, mRNA, siRNA, DNA, RNPs, proteins, and other cargos ○ Primary and patient-derived cell lines including immune and stem cells ● Cells are boosted through a membrane at low operating pressure (3-15 PSI) ○ Membrane format1 enables high-throughput cellular engineering A Drug Discovery Platform Enabling Robust Intracellular Delivery of Cell Impermeable Cargo via Mechanoporation Sophia Hirsch, Zhihui Song, Andrew Larocque, Rachel Conover, Athira Pillai, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA. www.portal.bio 1. Sharei, et al., PNAS, 2013 2. Szeto, et al., Scientific Reports, 2015 3. Sharei, et al., PLOS One, 2015 4. Lee and Sharei, et al. Nano Letters 2012 5. 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 macromolecules2,3,4 to both primary cells and cell lines ● Cells are 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 seals5. ○ Cells can be mixed with cargo or added before membrane re-sealing ### Summary ● Portal’s mechanoporation technology can be used for drug discovery and cell engineering ● Compatible across broad cell types including: NK cells HSCs B cellsPBMCs T cells iPSCs Connect with Portal! Contact us for beta program eligibility Mechanoporation Can be Used to Deliver Membrane Impermeable Cargo to Cells Improved delivery using mechanoporation Mixed PBMCs Cell impermeable small molecules Efficient Co-delivery of Antibody, Oligos, and Dextran to HeLa Cells Mixed isotype control antibody, labeled oligo, and fluorescent dextran Antibody Oligo Dextran Fluorescently-labeled IgG isotype control antibody, Cy5-labeled oligo, and fluorescent dextran were mixed and mechanoporated into HeLa cells. Flow cytometry was conducted post-boost to assess viability and delivery of the cargo types to the cells. Robust DNA Encoded Library Delivery in iPSCs and HeLa Cells iPSCs Fluorescent-tagged OR iPSC Boost HeLa Boost Induced pluripotent stem cells (iPSCs) or HeLa cells were mechanoporated with a fluorescently-tagged DNA encoded library (DEL) probe. Delivery efficiency was measured using flow cytometry to detect the fluorescent tag immediately after boosting. Boost with no cargo Pipette into plate pre-loaded with dextran ### N = 3 Delivery to HeLa Cells by Subsequent Cargo Addition 40s 5s 10s 20s 15s 25s 30s 35s 3 kDa dextran Enabling High Throughput Screening by Automated Dispensing into Pre-loaded Cargo Plates Boost with no cargo Dispense into 96 well plates pre-loaded with dextran 24h post-boost Enabling High Throughput Screening by Mixing Cargo Post-Boost Dextran Delivery to HeLa Cells by Automated Dispensing Well # (Plate 2) Well # (Plate 7) 10 min post-boost HeLa cells were boosted then manually added into wells of a 96-well plate pre-loaded with Alexa Fluor 488 3 kDa dextran at 5 second intervals. Flow cytometry was conducted post-boost to assess efficiency of dextran delivery into boosted cells. A cartridge was designed for integration of mechanoporation with a robotic single-head dispenser. HeLa cells were automatically dispensed into 96-well plates pre-loaded with fluorescent dextran. Flow cytometry was used to assess dextran delivery. Data for delivery across the 2nd (wells 97-192) and 7th (wells 577-672) 96-well plates are shown here. Phase contrast images (right) show morphology of HeLa cells 10 minutes (top) and 24 hours (bottom) post-boost. Scale bar (white) = 300 μm. A cell impermeable JAK-STAT pathway inhibitor was identified in a cell-free screen. This small molecule was delivered to mixed PBMCs using either electroporation (orange) or mechanoporation (blue). Flow cytometry was used to assess viability and delivery of the JAK compound into the cells (left). Activity of the inhibitor was confirmed by assessing knockdown of STAT in the mixed PBMCs (right). Li J, et al. 2017. ACS Chemical Biology. 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 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. Macrocycle Delivery to HeLa cells Shows Robust Dose Response Macrocycle Boost with no cargo Efficient Delivery of Labeled Macrocycle to HEK293 Cells HEK293 cells FITC-labeled macrocycle FITC Transmitted light HEK293 cells were mechanoporated with a FITC-labeled macrocycle. Flow cytometry was used to assess viability and macrocycle delivery to the untreated and boosted samples. Delivery was also assessed by fluorescence microscopy (right). Scale bar = 300 μm. Pipette into plate pre-loaded with labeled macrocycle HeLa cells were boosted then manually added into wells of a 96-well plate pre-loaded with FITC-labeled macrocycle at 0-0.2 mg/ml. Flow cytometry was conducted post-boost to assess viability and efficiency of macrocycle delivery into boosted cells. Sequential Delivery in Unstimulated and Activated T Cells Activated Sequential T Cell Boost with CRISPR RNP, mRNA Activate T cells ### Trac ### Rnp Activated T cells B2M expression TRAC expression B2M & TRAC Knockout & GFP Expressing Cells 51.4% Naive T cells were mechanoporated with T Cell Receptor Alpha Constant (TRAC) CRISPR RNPs. T cells were activated for 72h, then mechanoporated after 48h with a multiplexed cargo containing GFP mRNA and β2 microglobulin (B2M) CRISPR RNPs. After 40h, expression of TRAC protein, B2M protein, and GFP were assessed by flow cytometry. mRNA & B2M ### Rnp 1. Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999~ Efficient JAK Compound Delivery Corresponding STAT Knockdown www.portal.bio ● Can be used to deliver single or multiplexed cargoes ● Portal’s Research Scale Gateway™ enables complex cell engineering and delivery of cell impermeable molecules with a simple workflow ○ Small molecules, peptides, and proteins can be delivered into cells at scale for use in downstream screening ● Little impact on normal cellular function or morphology 0 μM 12 μM 24 μM 48 μM 96 μM ## 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.