Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases.

The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5(Δ32/Δ32)) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5(Δ32/Δ32) donors, we reasoned that engineered autologous CD34(+) hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.

Development and application of a multiplexable flow cytometry-based assay to quantify cell-mediated cytolysis.

Although target cell cytolysis has been widely employed to describe effector function of cells, cytolysis assays as commonly employed do not generate quantitative data. In this report we describe the development and application of a statistically supported flow cytometry-based assay to quantify cell-mediated cytolysis. The assay depends on the use of the fluorescent dye CFSE to distinguish target from effector cells, the DNA intercalating dye 7AAD to distinguish dead from live cell events, and on the establishment of a cytolysis curve that allows for the derivation of statistically robust data. We demonstrate that the cytolysis curve is well described by a four parameter logistic regression model provided that (i) the range of effector to target (E:T) ratios studied allows for full description of the logistic curve, and (ii) an adequate number of data points are collected to estimate the model parameters. We show that the assay is highly reproducible and accurate, and comparable in sensitivity with the standard (51)Cr assay. We report on the potential for this assay to generate quantitative data on the cytolytic activity of both CD8 T and NK cells; describe a relationship between the efficiency of effector cell degranulation and target cell cytolysis throughout a range of E:T ratios, and demonstrate the potential to multiplex with other platforms to obtain broader datasets for the effector phenotype of cells. Appropriate use of this assay will enhance the ability to derive quantitative and integrated correlative datasets from basic, translational, and clinical studies.

Optimized processing of growth factor mobilized peripheral blood CD34+ products by counterflow centrifugal elutriation.

Cell separation by counterflow centrifugal elutriation has been described for the preparation of monocytes for vaccine applications, but its use in other current good manufacturing practice (cGMP) operations has been limited. In this study, growth factor-mobilized peripheral blood progenitor cell products were collected from healthy donors and processed by elutriation using a commercial cell washing device. Fractions were collected for each product as per the manufacturer's instructions or using a modified protocol developed in our laboratory. Each fraction was analyzed for cell count, viability, and blood cell differential. Our data demonstrate that, using standard elutriation procedures, >99% of red blood cells and platelets were removed from apheresis products with high recoveries of total white blood cells and enrichment of CD34+ cells in two of five fractions. With modification of the basic protocol, we were able to collect all of the CD34+ cells in a single fraction. The CD34-enriched fractions were formulated, labeled with a ferromagnetic antibody to CD34, washed using the Elutra device, and transferred directly to a magnetic bead selection device for further purification. CD34+ cell purities from the column were extremely high (98.7 ± 0.9%), and yields were typical for the device (55.7 ± 12.3%). The processes were highly automated and closed from receipt of the apheresis product through formulation of target-enriched cell fractions. Thus, elutriation is a feasible method for the initial manipulations associated with primary blood cell therapy products and supports cGMP and current good tissue practice-compliant cell processing.