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 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. Bugge: 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.