# Rapid, Gentle, and Scalable Engineering of Hematopoietic Stem Cells using Vector-Free Microfluidic Cell Squeeze® Technology 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: Rapid, Gentle, and Scalable Engineering of Hematopoietic Stem Cells using Vector-Free Microfluidic Cell Squeeze® Technology - Document type: Poster - Year: 2022 - Outlet: ASGCT - Page URL: https://portal.bio/publications/2022-rapid-gentle-and-scalable-engineering-of-hematopoietic-stem-cells-using-vector-free-m--ajkGqBEAACgAc98F - Markdown URL: https://portal.bio/publications/2022-rapid-gentle-and-scalable-engineering-of-hematopoietic-stem-cells-using-vector-free-m--ajkGqBEAACgAc98F.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65ccddbc9be9a5b998b5d15f_FinalHSCPoster5.11.22.pdf ## Summary Murillo Silva, Jacquelyn Hanson, Jonathan Gilbert, and Devin T Bridgen ## Publication text Efficient delivery and expression of multiple mRNA targets in vitro and in vivo Cell Squeeze® technology efficiently delivers mRNA and RNPs - Greater than 90% of human HSCs express the transferred mRNA - Cell Squeeze treated HSCs can be transferred immediately following treatment, prolonging the expression window in-vivo. - High editing rates can be achieved in CD34+ cells when delivering anti-B2M and anti-CCR5 RNPs Cell Squeeze® technology maintains cell phenotype - Cell Squeezed treated CD34+ cells behave similarly to control cells as determined by microarray analysis, a CFC assay, and NBSGW mouse engraftment study. Electroporation induces functional changes in CD34+ cells - CD34+ cells exposed to electroporation have substantial changes in gene expression relative to squeezed cells - Electroporated cells plated in a CFC assay within two hours of treatment show an inability to form colonies at rates similar to the control - Electroporated cells transferred immediately after treatment engraft at lower rate and have altered differentiation capacity Cell Squeeze delivery is a desirable method for mRNA transfection or genetic engineering of primary human CD34+ cells - In combination with our point-of-care system under development, the ability to efficiently engineer HSCs as well as rapidly use the cells after manipulation, may enable novel HSC treatment paradigms closer to the patient. Edited BFU-E colony formation Colony formation Representative Images Day 0 Day 1 Thaw human donor CD34+ cells … Overnight Inject cells into NBSGW mice immediately after treatment 100k cells/animal Week 4-6 Peripheral blood Flow cytometry Control EPSqueeze Week 8 End of study Flow cytometry0 2 4 6 8 10 Peripheral blood chimerism Weeks Ctrl Squeeze EP **Ctrl Squeeze EP Spleen chimerism (wk8) ✱✱✱Ctrl Squeeze EP Bone marrow chimerism (wk8) ✱0 B2M Expression, Day 4 ### % B2m+ ### B2m Rnptide Ice Editing efficiency Genomic Analysis % Indels β2M RNP ### Ccr5 Rnp0 Viability % Live ### B2m Rnp Day 0 Day 4 Cell Squeeze ® β2MCtrl Squeeze EP Spleen Myeloid (wk8) %CD14+ of total human cells ✱✱Ctrl Squeeze EP Spleen Lymphoid (wk8) %CD19+ of total human cells ✱✱✱✱ ### Cxcr4gfp ### Gfp Cell Squeeze® Technology with differentiated capabilities Immune Cells RBCs HSC/iPSCs Gene editing complexes Proteins and peptides Nanomaterials Nucleic Acids Small molecules Robust across material classes Preservation of cell function Applicable to diverse cell types EP Squeeze Electroporation (EP) results in substantial gene misregulation vs. Squeezing Cells and target cargo together in suspension Cells are squeezed through SQZ chip at high speeds Cell membranes are temporarily disrupted Target cargo enters the cytosol of the cells Membranes reseal Rapid, Gentle, and Scalable Engineering of Hematopoietic Stem Cells using Vector-Free Microfluidic Cell Squeeze® Technology Murillo Silva1, Jacquelyn Hanson1, Jonathan Gilbert1, and Devin T Bridgen1 1SQZ Biotechnologies, Watertown, MA USA Introduction Efficient CRISPR editing of human HSCs Cell Squeeze® has minimal impact on phenotype and differentiation capacity Efficient engraftment of squeeze engineered HSCs immediately after treatment ### Summary HSCs Electroporation ### (ep) Gene expression changes are greater following electroporation than squeeze Cell Squeeze treated CD34+ cells form colonies at rates comparable to control HSCs β2M RNP Multi-plex genome editing with minimal effect on viability through Cell Squeeze® Day 0 Day 1 Human HSCs Overnight Immediately inject cells into NSG mice 100k cells/animal Bone Marrow Flow cytometry Ctrl Cell Squeeze® tech to introduce GFP mRNA Day -1 HSCs GFP or CXCR4 mRNA Cell Squeeze® technology does not impair HSC engraftment or cellular differentiation Treatment of numerous rare genetic diseases, including hemoglobinopathies, using gene edited autologous hematopoietic stem cells (HSCs) has shown potentially curative impact. However, effective engineering of HSCs, a sensitive cell type, is often limited by toxicity, cargo restrictions, and scalability challenges of current delivery methods. In preclinical studies, microfluidic deformation using the Cell Squeeze® technology effectively delivers diverse cargo into sensitive cell types and minimally alters the cell state and health of the cells. This is achieved through rapid and transient disruption of the cell membrane, which allows materials in solution around cells to diffuse into the cytosol. The Cell Squeeze® platform has been implemented in multiple clinical programs and reliably engineers billions of cells per minute. 24hr Comparison of bone marrow expression Efficient editing in resultant coloniesGFP Ctrl GFP expression % positiveGFP Ctrl GFP expression (in vivo) GFP (gMFI)CXCR4 mRNA CXCR4 expression Fold increase Expression of target gene in vivo in the bone marrow Efficient expression of GFP and surface receptor CXCR4 in vitro 6 hours Squeeze Electroporation5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8 -0.9 -1.0 -1.1 -1.2 -1.3 -1.4 -1.5 -1.6 -1.7 -1.8 -1.9 -2.0 -2.1 -2.2 -2.3 -2.4 -2.5 -2.6 -2.7 -2.8 -2.9 -3.0 -3.1 -3.2 -3.3 -3.4 -3.5 -3.6 -3.7 -3.8 -3.9 -4.0 -4.1 -4.2 -4.3 -4.4 -4.5 -4.6 -4.7 -4.8 -4.9-5.0 Log2 Fold change -5 Note: HSCs were treated and immediately plated Control EP Squeeze ### Gfp ## 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.