These steps include designing the optimal gRNA based on computational and mathematical predictions and the application of functional experimental off-target cleavage validation assays using GUIDE-seq8 and rhAmp-Seq10 technologies. therapy. Visual Abstract Open in a separate window Introduction Multivirus-specific T cells (VSTs) have proven safe and effective for the treatment of life-threatening viral infections such as cytomegalovirus (CMV), Epstein-Barr virus, adenovirus, and BK virus (BKV) after allogeneic hematopoietic stem cell transplant (HSCT).1 However, after HSCT, many patients receive steroids for the treatment of complications such as graft-versus-host disease (GVHD). Indeed, after HSCT, viral reactivation often follows the use of glucocorticoids. 2 Glucocorticoids are lymphocytotoxic and induce apoptosis of T cells, 3 thereby limiting the clinical efficacy of adoptively infused VSTs. Glucocorticoids exert their potent immunosuppressive effect by binding to the glucocorticoid receptor (GR), a pleiotropic ligand-activated transcription factor.4,5 Therefore, engineering VSTs to render them steroid resistant by deleting the nuclear receptor subfamily 3 group C member1 (knockout (KO) was performed on days 7 to 10 of VST expansion using the ribonucleoprotein (RNP) complex. We used 2 CRISPR RNAs (crRNAs) targeting exon 6-(γ,γ-Dimethylallylamino)purine 2 of the human gene: crRNA#1, TGA?GAA?GCG?ACA?GCC?AGT?GA; crRNA#2, GGC?CAG?ACT?GGC?ACC?AAC?GG. First, the crRNA plus (KO conditions). We used the Platinum SuperFi Green PCR Master Mix from Invitrogen for polymerase chain reaction (PCR) amplification using the following PCR primers spanning the Cas9Csingle-guide RNA cleavage site of exon 2 of the gene: exon 2 forward primer, GGA?CTC?CAA?AGA?ATC?ATT?AAC?TCC?TGG; exon 2 reverse primer, AAT?TAC?CCC?AGG?GGT?GCA?GA. DNA bands were separated by polyacrylamide gel electrophoresis prepared with SYBR-safe DNA gel stain in 0.5 Tris/Borate/EDTA. Gel images were obtained using a GBox machine with GeneSys software (Syngene). Band intensities were analyzed using Image J software by plotting the band intensities for each lane. The KO efficiency for (percentage) was calculated by dividing the intensity of the cleaved band by the intensity of the uncleaved control 6-(γ,γ-Dimethylallylamino)purine band. Western blot To detect GR protein expression, VSTs were lysed in lysis buffer (IP Lysis Buffer; Pierce Biotechnology Inc) supplemented with protease inhibitors (Complete Mini, EDTA-free Cocktail tablets; Roche Holding) and incubated for 30 minutes on ice. Protein concentrations were determined by the bicinchoninic acid assay (Pierce Biotechnology Inc). The following primary antibodies were used: GR (clone D6H2L) XP rabbit monoclonal antibody and -actin antibody (clone 8H10D10); both antibodies were obtained from Cell Signaling Technology. Blots were imaged using the LI-COR Odyssey Infrared Imaging System. Bands were quantified using Image J software. The percentage (%) of GR protein loss was calculated relative to values Mrc2 in control cells and normalized to -actin loading control. Flow cytometry The following antibodies 6-(γ,γ-Dimethylallylamino)purine were used for flow cytometry staining: anti-human CD3 antibody (BV650, clone UCHT1; BD Biosciences), anti-human CD4 antibody (allophycocyanin [APC], clone RPA-T4; Invitrogen), anti-human CD8 antibody (peridinin chlorophyll protein [Percp], clone SK1; BioLegend), anti-human CD62L (BV605, clone DREG-56; BD Biosciences), anti-human CD45RA (phycoerythrin-Cy7, clone HI100; BD Biosciences), and anti-human CCR7 (fluorescein isothiocyanate, clone G03H7; BioLegend). All data were acquired with BD Fortessa (BD Biosciences) and analyzed with FlowJo software. Annexin V apoptosis assay To evaluate the effect of dexamethasone on the viability of VSTs (Cas9 control or KO), the annexin V apoptosis assay was performed. Cas9 control or KO VSTs were treated with dexamethasone (200 M; Sigma) for 72 hours, the cells were collected and washed with annexin V buffer and stained with annexin V (V500; BD Biosciences) and live/dead (efluor 660; Invitrogen) in addition to CD3 (BV650, clone UCHT1; BD Biosciences), CD4 (APC, RPA-T4; Invitrogen), and CD8 (Percp, clone SK1; BioLegend). 6-(γ,γ-Dimethylallylamino)purine After gating on T cells, the proportion of apoptotic (positive for annexin V) and dead cells (positive for live/dead stain) was determined by flow cytometry..
