# Non-genotoxic conditioning approach for autologous hematopoietic stem cell therapies through transient mRNA engineering 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: Non-genotoxic conditioning approach for autologous hematopoietic stem cell therapies through transient mRNA engineering using vector-free microfluidic Cell Squeeze technology
- Document type: Poster
- Year: 2022
- Outlet: ASH
- Cells: CD34+
- Cargos: Polymers, Protein, Peptide, mRNA, CRISPR RNPs
- Page URL: https://portal.bio/publications/2022-non-genotoxic-conditioning-approach-for-autologous-hematopoietic-stem-cell-therapies--ajkGcREAAC0Ac95c
- Markdown URL: https://portal.bio/publications/2022-non-genotoxic-conditioning-approach-for-autologous-hematopoietic-stem-cell-therapies--ajkGcREAAC0Ac95c.md
- Original file: https://portal-bio.cdn.prismic.io/portal-bio/65cbeb749be9a5b998b5c740_2022.11.16_ASHPoster-VF.pdf

## Summary

Murillo Silva, Cara Bartlett, Jacquelyn Hanson, Jonathan Gilbert, and Devin T Bridgen

## Publication text

Cell Squeeze® technology efficiently delivers mRNA

- 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. Cell Squeeze® technology maintains cell phenotype better than electroporation

- Cell Squeeze® treated HSCs behave similarly to control cells as determined by microarray

analysis, a CFC assay, and NBSGW mouse engraftment study.

- HSCs exposed to electroporation have substantial changes in gene expression, CFC

assay and in-vivo engraftment. Cell Squeeze® delivery of Plerixafor-resistant CXCR4 mRNA provides a competitive homing advantage to engineered HSCs

- HSCs expressing CXCR4 (A175F) mRNA maintain CXCL12 induced signaling in the

presence of Plerixafor. This strategy could be used to promote engraftment of adoptively transferred cells. Cell Squeeze® delivery of Venetoclax-resistant BCL2 mRNA provides a competitive survival advantage to engineered HSCs

- HSCs expressing BCL2 (G101V) remain viable in the presence of Venetoclax. This

strategy could be used to promote a competitive survival advantage to adoptively transferred cells vs endogenous HSCs. Goal: Improve HSC transplants for non-malignant diseases 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 Cell Squeeze® Technology has minimal impact on CD34+ phenotype and differentiation capacity Approach 1: Engineering a competitive homing advantage with CXCR4 in adoptively transferred HSCs Summary0 100000 200000 300000 Hours post treatment BCL2 Expression (gMFI)

### Ctrl

BCL2 (wt) BCL2 (G101v) 4 24 48 720 24 48 0.00 0.25 0.50 0.75 1.00 Hours post treatment Cells Remaining (normalized to time 0)

### Ctrl

BCL2 (wt)

### Bcl2 (g101v)

Mutant BCL2 (G101V) enables resistance to Venetoclax apoptosis Label with CellTrace® Violet or Far RedGFP Transfered cell frequency (% of human cells) nsGFP Transfered cell frequency (% of human cells) ✱✱✱✱ Approach 2: Engineering a competitive survival advantage with BCL2 in adoptively transferred HSCs Mutant BCL2 (G101V) overexpression promotes HSC survival in the presence of Venetoclax in NSG mice Cell Squeeze® Technology Overview Applicable to diverse cell types Preservation of cell function Scalable and clinically implemented Colony formation Edited BFU-E colony formation 6 hours Post-delivery5.0 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 Gene expression changes 6hr after delivery Gene expression and colony formation after squeezing is comparable to control in contrast to electroporation (EP) Efficient Engraftment of Squeeze Engineered HSCs Immediately After Treatment Day 0 Day 1 Thaw human donor CD34+ cells … Overnight culture 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) ns ✱✱✱Ctrl Squeeze EP Bone marrow chimerism (wk8) ns ✱0 20 40 60 80 Plerixafor + CXCR4 A175F mRNA Plerixafor + CXCR4 mRNA Plerixafor alone Plerixafor mediated CXCR4 signaling inhibition % Reduction in Ca2+ flux (normalized to CXCL12 w/o Plerixafor)0 20 40 60 80 100 NC post-squeeze viability (%)0 10000 20000 30000 NC CXCR4 expression on LT-HSCs 24hr post-squeeze (gMFI) Cell Squeeze® enables efficient CXCR4 mRNA expression Mutant CXCR4 (A175F) expression enables resistance to Plerixafor mediated inhibition Representation of Approach with Plerixafor Resistant CXCR4 Expression BCL2 overexpression after mutant (G101V) or wild type (wt) BCL2 mRNA delivery Day 0 Day 1 Overnight culture Day 2 Bone-marrow flow cytometry

### Gfp

### Bcl2g101v

mRNA Transfer to NSG mice Thaw human donor CD34+ cells Dose w/ Venetoclax & S63845 No Treatment Venetoclax Overview Therapeutic treatment options for hemoglobinopathies as well as many other monogenic diseases, are typically non-curative and have significant limitations. Autologous gene edited Hematopoietic Stem Cell (HSC) transplant has demonstrated potentially curative effects in recent clinical trials but is limited by low engraftment efficiencies and significant toxicities associated with the required myeloablative preconditioning agents. Our overall objective is to establish a safer and more effective autologous transplant method using mRNA enhanced HSCs and a modified conditioning regimen that is non-genotoxic. We will use the Cell Squeeze® technology to engineer HSCs ex vivo with mRNA to transiently outcompete the endogenous diseased HSCs. To create HSCs with potentially enhanced engraftment, we delivered mRNA constructs encoding the homing receptor CXCR4 or survival factor BCL2. Furthermore, to provide a competitive advantage against endogenous HSCs, we tested mRNA sequences with selected mutations to create cells resistant to conditioning agents Plerixafor, a CXCR4 antagonist, and Venetoclax, a BCL2 inhibitor. By transiently expressing the engraftment factors, the constructs are no longer expressed after a few days, and the HSCs could be maintained naturally in the now occupied niche. Current: Engineer the patient with toxic chemotherapy Future: Engineer the cell for enhanced engraftment Mutant BCL2 promotes survival in vivo under Venetoclax pressure Expression of target proteins by transferred HSCs in the bone marrow at 24hrsGFP 10000 20000 30000 40000 BCL2 expression (gMFI) Squeezed with

### Bcl2 (g101v)

Squeezed with GFP

### Gfp

### Bcl2

Non-Genotoxic Conditioning Approach for Autologous Hematopoietic Stem Cell Therapies through Transient mRNA Engineering using Vector-Free Microfluidic Cell Squeeze® Technology Murillo Silva1, Cara Bartlett1, Jacquelyn Hanson1, Jonathan Gilbert1, and Devin T Bridgen1 1SQZ Biotechnologies, Watertown, MA USAGFP 20000 40000 60000 GFP expression (gMFI)

## 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.