Poster · 2024 · ISSCR: International Society for Stem Cell Research

A platform for direct cytosolic delivery via mechanoporation: opening opportunities for multiplexed and sequential deliveries accelerating cell reprogramming and differentiation

Sophia Hirsch, Zhihui Song, Rachel Conover, Darby Kreienberg, Athira Pillai, Andrew Larocque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei

Cells: T cells, PBMCs, iPSCs, CD34+
Cargos: mRNA, CRISPR RNPs, DELs, siRNA, circRNA

Publication text

Induced pluripotent stem cells (iPSCs) were mechanoporated and cultured post-boost. After 48h, total RNA was extracted from cells and RT-qPCR was conducted on untreated and boosted cells to assess endogenous control gene expression (left) and pluripotency markers (right). Data were analyzed for two independent experiments. Research Scale Gateway™ High Throughput Compact bench-top unit that controls delivery parameters and easily fits in hood Introduction to Portal’s Approach: Implementing A Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo

Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999

● Over a billion cells in under a minute ● “Powered by Portal” consumables Clinical Scale Integrate with 3rd Party manufacturing equipment ● Portal has created a cell engineering technology compatible across cell types and materials ○ Primary and patient-derived cell lines including immune and stem cells ○ mRNA, siRNA, cRNA, saRNA, DNA, RNPs, proteins, and other cargos ● Cells are boosted through a membrane at low operating pressure (<1 bar) ○ Membrane format1 enables high-throughput cellular engineering A platform for direct cytosolic delivery via mechanoporation: opening opportunities for multiplexed and sequential deliveries accelerating cell reprogramming and differentiation Sophia Hirsch, Zhihui Song, Rachel Conover, Darby Kreienberg, Athira Pillai, Andrew Larocque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA

Summary

● Portal’s mechanoporation technology can be used for genetic and/or RNA-based cell engineering ○ Compatible across broad cell types including: ○ Can be used to deliver single 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 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 Connect with Portal! Contact us for beta program eligibility info@portal.bio ● Integrates with existing liquid handlers ● Ready for early adopters late 2024 “Powered by Portal” Consumables NK cells HSCs PBMCs T cells Multiplex Delivery in Unstimulated T Cells T Cell Boost with CRISPR RNP, mRNA, and Dextran 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. High Knock Down Efficiency in CD34+ Cells Hematopoietic Stem Cell (HSC) boost with siRNA CD34+ cells B2M expression 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. Efficient mRNA Expression in Human iPSCs and MSCs iPSCs and MSCs boosted with GFP mRNA Induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs) were mechanoporated with GFP mRNA. After 20h, GFP expression in iPSCs was assessed using fluorescence microscopy and flow cytometry. GFP expression in MSCs was assessed using flow cytometry and cell morphology was assessed by microscopy. Size (microns) refers to pore size used for mechanoporation. Enabling High Throughput Screening by Mixing Cargo Post Boost Boost with no cargo Pipette into plate pre-loaded with dextran 3 kDa dextran

N = 3

33s 11s 15s 20s 18s 23s 26s 29s Delivery to iPSCs by Subsequent Cargo Addition Efficient Multiplexed Delivery in Human iPSCs Induced pluripotent stem cell (iPSC) boost with GFP mRNA + B2M siRNA iPSCs were mechanoporated with GFP mRNA and β2 microglobulin (B2M) siRNA. After 40h, GFP and B2M expression were assessed by flow cytometry. iPSCs Maintain Expression of Housekeeping and Pluripotency Genes Post-Boost DNA Encoded Library Delivery in iPSCs DEL probe iPSC Boost siRNA iPSCs 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. GFP expression 10 μm 11 μm GFP Transmitted light Boost Untreated iPSCs were mechanoporated and subsequently added to a 96-well plate pre-loaded with fluorescent dextran. Dextran delivery was assessed by flow cytometry. Time is indicated in seconds post-boost. Circular RNA Expression in Mixed PBMCs Day 3 mRNA cRNAGFP mRNA GFP cRNA

-or-

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. Fluorescent-tagged DEL probe iPSCs Ascl1 saRNA+ 5TFs mRNA siRNA MSCs Mixed PBMCs B cells

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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.

# A platform for direct cytosolic delivery via mechanoporation: opening opportunities for multiplexed and sequential deliveries accelerating cell reprogramming and differentiation 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 direct cytosolic delivery via mechanoporation: opening opportunities for multiplexed and sequential deliveries accelerating cell reprogramming and differentiation - Document type: Poster - Year: 2024 - Outlet: ISSCR: International Society for Stem Cell Research - Cells: T cells, PBMCs, iPSCs, CD34+ - Cargos: mRNA, CRISPR RNPs, DELs, siRNA, circRNA - Page URL: https://portal.bio/publications/2024-a-platform-for-direct-cytosolic-delivery-via-mechanoporation-opening-opportunities-fo--ajkGVxEAACoAc93_ - Markdown URL: https://portal.bio/publications/2024-a-platform-for-direct-cytosolic-delivery-via-mechanoporation-opening-opportunities-fo--ajkGVxEAACoAc93_.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFmpj3fc4bHWiobl_ISSCRposter5JUL24.pdf ## Summary Sophia Hirsch, Zhihui Song, Rachel Conover, Darby Kreienberg, Athira Pillai, Andrew Larocque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei ## Publication text Induced pluripotent stem cells (iPSCs) were mechanoporated and cultured post-boost. After 48h, total RNA was extracted from cells and RT-qPCR was conducted on untreated and boosted cells to assess endogenous control gene expression (left) and pluripotency markers (right). Data were analyzed for two independent experiments. Research Scale Gateway™ High Throughput Compact bench-top unit that controls delivery parameters and easily fits in hood Introduction to Portal’s Approach: Implementing A Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo 1. Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999 ● Over a billion cells in under a minute ● “Powered by Portal” consumables Clinical Scale Integrate with 3rd Party manufacturing equipment ● Portal has created a cell engineering technology compatible across cell types and materials ○ Primary and patient-derived cell lines including immune and stem cells ○ mRNA, siRNA, cRNA, saRNA, DNA, RNPs, proteins, and other cargos ● Cells are boosted through a membrane at low operating pressure (<1 bar) ○ Membrane format1 enables high-throughput cellular engineering A platform for direct cytosolic delivery via mechanoporation: opening opportunities for multiplexed and sequential deliveries accelerating cell reprogramming and differentiation Sophia Hirsch, Zhihui Song, Rachel Conover, Darby Kreienberg, Athira Pillai, Andrew Larocque, Jacquelyn Hanson, Alec Barclay, Mathias Pawlak, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA ### Summary ● Portal’s mechanoporation technology can be used for genetic and/or RNA-based cell engineering ○ Compatible across broad cell types including: ○ Can be used to deliver single 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 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 Connect with Portal! Contact us for beta program eligibility info@portal.bio ● Integrates with existing liquid handlers ● Ready for early adopters late 2024 “Powered by Portal” Consumables NK cells HSCs PBMCs T cells Multiplex Delivery in Unstimulated T Cells T Cell Boost with CRISPR RNP, mRNA, and Dextran 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. High Knock Down Efficiency in CD34+ Cells Hematopoietic Stem Cell (HSC) boost with siRNA CD34+ cells B2M expression 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. Efficient mRNA Expression in Human iPSCs and MSCs iPSCs and MSCs boosted with GFP mRNA Induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs) were mechanoporated with GFP mRNA. After 20h, GFP expression in iPSCs was assessed using fluorescence microscopy and flow cytometry. GFP expression in MSCs was assessed using flow cytometry and cell morphology was assessed by microscopy. Size (microns) refers to pore size used for mechanoporation. Enabling High Throughput Screening by Mixing Cargo Post Boost Boost with no cargo Pipette into plate pre-loaded with dextran 3 kDa dextran ### N = 3 33s 11s 15s 20s 18s 23s 26s 29s Delivery to iPSCs by Subsequent Cargo Addition Efficient Multiplexed Delivery in Human iPSCs Induced pluripotent stem cell (iPSC) boost with GFP mRNA + B2M siRNA iPSCs were mechanoporated with GFP mRNA and β2 microglobulin (B2M) siRNA. After 40h, GFP and B2M expression were assessed by flow cytometry. iPSCs Maintain Expression of Housekeeping and Pluripotency Genes Post-Boost DNA Encoded Library Delivery in iPSCs DEL probe iPSC Boost siRNA iPSCs 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. GFP expression 10 μm 11 μm GFP Transmitted light Boost Untreated iPSCs were mechanoporated and subsequently added to a 96-well plate pre-loaded with fluorescent dextran. Dextran delivery was assessed by flow cytometry. Time is indicated in seconds post-boost. Circular RNA Expression in Mixed PBMCs Day 3 mRNA cRNAGFP mRNA GFP cRNA ### -or- 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. Fluorescent-tagged DEL probe iPSCs Ascl1 saRNA+ 5TFs mRNA siRNA MSCs Mixed PBMCs B cells ## 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.