Vector Free Genome Editing of Human CD34+ Cells for Cell Therapy

Abstract

While the ex vivo manipulation of primary cells has signaled a new era in the application of cell-based therapies, common methods to manipulate primary cells have limitations. To overcome the limitations associated with conventional cell delivery and engineering systems, we have developed an approach to delivery where cells are mechanically deformed as they pass through a constriction. This cellular deformation results in the diffusion of material from the surrounding buffer directly into the cytosol. This system has demonstrated efficacy in patient-derived cells, such as stem cells and immune cells, with a variety of target molecules that are difficult to address with alternative methods. Moreover, by eliminating the need for electrical fields or exogenous materials such as viral vectors and plasmids, it minimizes the potential for cell toxicity and off-target effects. Here, we present evidence detailing our ability to deliver functional material to primary human CD34+ cells via cell deformation with little detectable perturbation in baseline gene expression, cell function, and viability. To determine effect of cell deformation on gene expression and to compare to other delivery systems, human CD34+ cells (n = 3 donors) were subjected to cell deformation or electroporation and gene expression changes were compared to unmanipulated control cells using microarray analysis. Differential gene expression with respect to both methods of delivery was assessed by performing t tests on the coefficient of a linear mixed-effects model that treated delivery method as a fixed effect and donor as a random effect. Electroporation produced substantially more changes in gene expression (5,285 genes with FDR q < 0.25) than cell deformation (no genes with FDR q < 0.25) as compared to untreated controls. Subsequently, we designed a series of experiments to manipulate gene expression with the CRISPR-CAS9 system using cell deformation to deliver CAS9 ribonucleoproteins (RNPs; recombinant CAS9 protein complexed with a single-guide RNA) designed to edit a model locus, the C-C chemokine receptor type 5 (CCR5). Here, we show that the delivery of the CRISPR-CAS9 system via cell deformation results in significant CCR5 mutagenesis. Furthermore, CD34+ cells subjected to cell deformation proliferate and differentiate at rates similar to unmanipulated control cells, as determined by the Colony-Forming Cell (CFC) assay. Disruption of the CCR5 locus was observed in individual BFU-E colonies by performing Sanger sequencing. These data suggest that cell deformation is a viable delivery method for genetic engineering of primary human CD34+ cells with little impact on baseline gene expression or the ability of hematopoietic progenitors to proliferate and differentiate. The ability to deliver structurally diverse materials to difficult-to-transfect primary CD34+ cells indicate that this method could potentially enable many novel clinical applications. Disclosures Bridgen: SQZ Biotechnologies: Employment, Equity Ownership. DiTommaso:SQZ Biotechnologies: Employment, Equity Ownership. Buggé:SQZ Biotechnologies: Employment, Equity Ownership. Gilbert:SQZ Biotechnologies: Employment, Equity Ownership. Bernstein:SQZ Biotechnologies: Employment, Equity Ownership. Sharei:SQZ Biotechnologies: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Delivering the CRISPR/Cas9 system for engineering gene therapies: Recent cargo and delivery approaches for clinical translation

“…Similarly, cell-squeezing devices with progressively narrow channels either 40–4 µm or 60–6 µm in diameter were shown to effectively deliver functional RNPs targeting the GFP gene in a stably-expressing GFP reporter U20S cell line, inducing ∼40% knockout of the GFP gene ( Uvizl et al, 2021 ) ( Figure 2B ). Similarly, Bridgen et al (2017) demonstated the ability to use cell squeezing in the delivery of Cas9 RNPs to primary human T cells and CD34 + hematopoietic stem and progenitor cells to edit the CCR5 and B2M loci with little detectable impact on cell differentiation, proliferation, and function, further establishing cell squeezing as a viable method for cell therapy manufacturing.…”

“…There are several approaches to temporarily porate the cell membrane, each with their own advantages and challenges. Methods to physically generate pores in the cell membrane via mechanoporation techniques, including microinjection ( Crispo et al, 2015 ; Hruscha and Schmid, 2015 ; Martin-Martin et al, 2018 ), microfluidics/cell squeezing ( Saung et al, 2016 ; Bridgen et al, 2017 ), and sonoporation ( Helfield et al, 2016 ) are all currently under development. The most widespread delivery method is electroporation, whereby cells are exposed to an electrical field in order to create pores in the membrane that facilitate reproducible and efficient intracellular entry of biomolecules into cells ( Deng et al, 2018 ; Kang et al, 2020 ).…”