HSPC were isolated from granulocyte-CSF–mobilized peripheral blood from healthy donors and purchased from vendors using previously described methods.13
Briefly, the washed and concentrated mobilized peripheral blood were labeled with CliniMACS CD34 microbeads (Miltenyi Biotec) and enriched with the CliniMACS Cell Separation System (Miltenyi Biotec). The purity of cells was usually >98%. Enriched CD34+
cells were either used freshly for transduction or cryopreserved using a controlled rate freezer and stored in the vapor phase of a liquid nitrogen dewar for later use.
The Ad5/F35-pseudotyped CCR5-ZFN (CCR5-ZFN) and Ad5/F35-pseudotyped GFP (Ad-GFP) used in this study were provided by Sangamo BioSciences (Richmond, CA) and described previously (Perez et al.11
and Nilsson, M. et al.29
). Briefly, CCR5-ZFN and Ad-GFP adenoviral vectors were generated on an E1/E3-deleted backbone. The pair of CCR5 ZFNs were linked via
a 2A-peptide sequence and cloned into the pAdEasy-1/F35 vector under control of the CMV TetO promoter (24). The Ad5/F35 virus for each construct was generated using TREx 293T cells as described.29
The vector was tested negative for the presence of replication-competent adenovirus before use in the study.
Adenoviral transduction of HSPCs and in vitro culture.
Freshly isolated or frozen CD34+
HSPC cells (1
cells/ml) were prestimulated in CellGro SCGM (CellGenix, Freiburg, Germany) supplemented with three cytokines (SFT): 100
ng/ml recombinant human SCF, 100
ng/ml recombinant human Flt-3L (Invitroge, Carlsbad, CA), and 10
ng/ml recombinant human TPO (CellGenix) 12–24 hours before transduction. After overnight prestimulation, cells were treated with PMA (Sigma-Aldrich, St Louis, MO) or bryostatin 1 (bryostatin) (Sigma-Aldrich) at indicated concentrations for 30 minutes and washed before transduction. Treated cells were suspended to a density of 1
cells/ml. Vectors were added to the cells at MOI of 0–5,000 for 12–24 hours and incubated at 37 °C in 5% CO2
. For myeloid hematopoietic potential, cells were incubated in a bulk culture medium (Iscove's modified Dulbecco's medium, 20% fetal bovine serum, 1% L
ng/ml of SCF, 100
ng/ml Flt-3L, 10
ng/ml of TPO, 40
ng/ml of IL-6, 20
ng/ml of IL-3, 3 IU/ml erythropoietin, and 100
ng/ml granulocyte macrophage-CSF) for up to 4 weeks.13
Samples were collected at the time points indicated for cell counts using Guava PCA-96 (EMD Millipore, Danvers, MA) and performance of other assays as listed below.
Colony-forming unit assay. Mock-transduced and PMA-treated, CCR5-ZFN–transduced CD34+ HSPCs were harvested 16–24 hours after transduction. A total of 500 cells were plated in triplicate in MethoCult H4435-enriched methylcellulose medium according to the manufacturer's instruction (Stem Cell Technologies, Vancouver, British Columbia, Canada); 12–14 days after incubation, total colonies were enumerated under inverted microscope.
Cell viability-apoptosis analysis by annexin V/7-AAD staining. Aliquots of control and transduced cells were stained with Annexin V FITC Apoptosis Detection Kit (BD Pharmingen, San Jose, CA) in Annexin-binding buffer (BD Pharmingen) as instructed by the manufacturer and phenotypic data was collected on an Cytomics FC500 flow cytometer (Beckman Coulter, Brea, CA) and analyzed with the FCS Express Version 3 software (DeNovo Software, Los Angeles, CA). Viable cells are the population that is negative for both annexin V and 7-AAD staining and are compared with the unstained control.
Surveyor nuclease assay. The percentage of CCR5 alleles disrupted by ZFN treatment was measured by surveyor nuclease assay. Briefly, genomic DNA was extracted using the MasterPure Complete Purification kit (Epicentre, Madison, WI) according to the manufacturer's instructions. The purified genomic DNA was used as a template to amplify a fragment of the CCR5 gene using the primers C5_Cel_160_F1: AAGATGGATTATCAAGTGTCAAGTCC; and C5_Cel_160_R1: CAAAGTCCCACTGGGCG in the presence of a 32P-dATP and dCTP. The PCR products were then heated, allowed to re-anneal followed by treatment with the mismatch-sensitive surveyor nuclease (Transgenomic, Omaha, NE) as described in order to detect insertions and deletions caused by NHEJ. The resulting radiolabeled products digested with the surveyor nuclease were resolved by polyacrylamide gel electrophoresis, and the ratio of cleaved to uncleaved products was calculated to give a measure of the frequency of gene disruption. The assay is sensitive enough to detect single-nucleotide changes.
Flow cytometric analysis. Aliquots of cells from in vitro culture were taken weekly for phenotypic analysis using antibodies to lineage-specific cell surface antigens. Antibodies CD33-PE, CD15-FITC, GlyA-PC5, (BD Biosciences, San Jose, CA), and CD14-APC-Alexa750 (Invitrogen, Carlsbad, CA) were used to identify for progenitors, granulocyte, erythyrocyte, and monocyte subpopulations, respectively. Phenotypic data were collected on a Gallios flow cytometer (Beckman Coulter) and analyzed with FCS Express Version 3 software (DeNovo Software). Lineage-specific subpopulations, CD14+, CD15+, CD14+/CD15+, and CD14−/CD15− cells from PMA-treated and CCR5-ZFN–transduced cells were sorted by fluorescence-activated cell sorting after 4 weeks culture in the bulk culture media that promotes myeloid-erythroid lineage differentiation.
Genotyping for single and biallele CCR5 knockout.
Mock-transduced and CCR5-ZFN–transduced CD34+
HSPC cells were harvested 16–24 hours after transduction and plated in triplicate in MethoCult H4435-enriched methylcellulose medium as described above in colony-forming unit assay. Individual colonies were isolated and analyzed for single and biallele CCR5 knockout by first screening for unmodified colonies using either the surveyor nuclease assay or a second PCR-based assay capable of detecting mutations at the CCR5-ZFN cleavage site using primers designed to specifically amplify unmodified wild-type sequences but not sequences with deletions, as previously described.30
These assays identified 160 methylcellulose colonies for further evaluation. The CCR5 locus was PCR-amplified from these cells, cloned into a bacterial plasmid, and then genotyped by directly sequencing the plasmid inserts.
Off-target site analysis.
cells were prestimulated in SCGM medium with SCF, Flt-3L, and TPO at 37 °C in 5% CO2
overnight. On the next day, cells were treated with 1
ng/ml PMA for 30 minutes. Cells were washed and transduced with Ad5/35 CCR5-ZFN at MOI of 10, and then cultured for 7 days before harvesting and isolating genomic DNA for initial PCR for the target loci followed by illumina deep sequencing. Briefly, initial PCR products were purified, treated with Klenow fragment (3′-5′ exo-; New England BioLabs, Ipswich, MA), ligated to adaptors, gel-purified, and then PCR-amplified (14 cycles) with illumina primers. Deep sequencing was performed on the top 23 off-target sites. The original top 15 potential off-target sites identified by a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol-guided analysis of the human genome; and eight additional sites obtained by an unbiased approach to off-target sites identification via
mapping the locations of integrase-defective lentiviral integration in CCR5-ZFN–treated cells by linear amplification-mediated PCR.17
cells were stained with phosphoprotein-specific monoclonal antibody as previously described.31
cells cultured in SCGM medium with three cytokines at 37 °C in 5% CO2
were harvested after 15 minutes of treatment with either PMA or bryostatin or mock-treated in a suitable amount of dimethyl sulfoxide containing medium. Cells were fixed with an equal volume of BD PhosFlow Fix Buffer I (BD Biosciences) for 10 minutes at 37 °C to arrest signaling activity and washed with staining buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin) followed by permeabilization in 90% cold methanol at 4 °C for 30 minutes, washed twice with staining buffer and split evenly for parallel staining with anti-STAT3 (pY705)-PE, anti-ERK1/2 (pT202/pY204)-PE, anti-STAT5 (pY694)-PE, anti-β-catenin (pS45)-PE, anti-Akt (pS473)–Alexa Fluor 647, anti-p38 (pT180/pY182)-PE, anti-NF-κBp65(pS529)-PE, and isotype controls (BD Biosciences) for 30 minutes at room temperature. After incubation with antibody, cells were washed with staining buffer, pelleted, and resuspended in phosphate-buffered saline buffer containing 0.1% bovine serum albumin for flow cytometric analysis. At least 50,000 events from each sample were collected using Cytomics FC500 flow cytometer (Beckman Coulter). Cells were gated on live population (clearly demarcated in forward/side scatter plots) for analysis of geometric mean of fluorescence intensity using FCS Express 3.0 software (Denovo Software).
RNA preparation and quantitative reverse transcription-PCR.
The effect of PMA on the expression of ZFN (Fok1) mRNA was examined using reverse transcription-PCR. CD34+
cells were seeded in six-well plates and supplemented with SCGM containing SCF, TPO, and Flt-3L as described above. One day after seeding, cells were stimulated with or without PMA (1
ng/ml) for 30 minutes. Then cells were washed, transduced with CCR5-ZFN (MOI 50), and cultured in SCGM with three cytokines for an additional 3 hours. Total RNA was extracted from untreated control; CCR5-ZFN–transduced; and PMA-treated, CCR5-ZFN–transduced CD34+
cells, with RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer's manual. Approximately 1 μg RNA was used for cDNA synthesis using the RT2
First strand kit (Qiagen) following the manufacturer's instruction. The following primers were used for amplification; Fok1 forward 5′-CCTGACGGCGCCATCTAT-3′ and reverse 5′-CGATCACGCCGTAATCGAT-3′, GAPDH forward 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse 5′-GAAGATGGTGATGGGATTTC-3′. Quantitative PCR measurements were performed on five biological replicates, PCR-amplified as three technical replicates and plotted as fold increase of ZFN-Fok1 RNA expression. Changes in the cycle threshold (ΔCT
) values for Fok1 gene expression were obtained by subtracting the mean threshold cycle (CT
) of the housekeeping gene (GAPDH) from the threshold cycle value of the Fok1 gene. Changes in the ΔCT
values for Fok1 gene were obtained by subtracting the mean CT
of the housekeeping gene (GAPDH) from the CT
value of the Fok1 gene. The ΔΔCT
value for five experiments was calculated using formula ΔΔCT
(treatment) − ΔCT
(untransduced control). The fold upregulation of Fok1 transcription was calculated as follows: 2(−ΔΔCT)
Western blot analysis.
cells/ml) were treated with bryostatin or PMA in presence or absence of GSK1120212 inhibitor (Selleck Chemicals, Houston, TX) for 30 minutes followed by transduction of CCR5-ZFN at MOI of 50 overnight (12–24 hours). Total cell lysates were prepared in a RIPA Lysis Buffer system (Santa Cruz Biotechnology, Santa Cruz, CA). The protein content of the samples was measured using the BCA protein assay reagent (Thermo Scientific, Pierce Protein Research Products, Rockford, IL). A total of 20 or 50 μg protein samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis using 10% Tris-HCl gels and blot transferred onto a polyvinylidene difluotide membrane, immunoblotted with anti-Fok1 antibody (Sangamo BioSciences) anti-ERK1/2, phospho-ERK1/2, IkBa, phospho-IkBa (Cell Signaling Technology, Danvers, MA) or β-actin (Sigma-Aldrich) antibodies. The bands were detected using the SuperSignal West Pico chemiluminescent detection system (Thermo Scientific, Pierce Protein Research Products).
NSG mouse transplantation and engraftment analysis.
NOD.Cg-Prkdc scid Il2rg tm1Wj/SzJ (NSG) mice were originally obtained from Jackson Laboratories (Bar Harbor, ME). Adult mice within 7–10 weeks of age received 250 cGy radiation, then either immediately or 2–4 hours later mice were retro-orbitally injected with 1
CCR-ZFN–modified or mock-treated human CD34+
HSPCs in 50 μl phosphate-buffered saline containing 1% heparin. Peripheral blood samples were collected every month starting at 4 weeks postengraftment using retro-orbital sampling. Whole blood was blocked in fetal bovine serum (Mediatech, Manassas, VA) for 30 minutes. The RBCs were lysed using Pharmlyse solution (BD Biosciences), and cells were washed with phosphate-buffered saline. Mice were necropsied between 8 and 28 weeks post-transplantation and tissues (spleen, BM) were collected and processed either immediately for cell isolation and fluorescence-activated cell sorting analysis as previously described by Holt and colleagues12
or spun down and frozen for later DNA extraction and surveyor nuclease analysis.
Fluorescence-activated cell sorting analysis of human cells was performed using a FACS Cyan instrument (BD Biosciences) with FlowJo software version 7.6.5 for PC (TreeStar). The initial gating strategy performed was based on forward scatter versus side scatter gate to exclude debris, followed by a human CD45 gate. Specific lineages (B cell, CD4+ T cell, CD8+ T cell, and monocytes) were determined using specific antibodies, with monocytes defined as CD4dim. All antibodies (BD Biosciences) were fluorochrome-conjugated and human-specific. The clones used in the studies are: CD45PerCP Cy5.5, clone 2D1; CD19APC, clone HIB19; CD4FITC, clone SK3; CD8PE, clone HIT8a.
HIV-1 challenge and CCR5 disruption analysis of NSG mice.
NSG mice were transplanted/engrafted with either untreated or PMA/CCR5-ZFN–modified HSPCs as described in the text. At 26 weeks of transplant, half of the cohort was necropsied and analyzed for % CD45 and CCR5 disruption in the BM and spleen. Of the remaining animals, five mice were infected with a cell-free virus stock of HIV-1BaL
that was propagated and administered as previously described.12
Eleven weeks post-infection, tissue samples were collected at necropsy and processed immediately for cell isolation and fluorescence-activated cell sorting analysis, or kept in freezing media for later analysis and DNA extraction. CCR5 disruption were analyzed using the Surveyor nuclease assay on bulk unsorted spleen cells and CD4+
, and CD19+
CCR5-ZFN transduction resulted in low level CCR5 disruption in HSPC CD34+
cells under standard conditions.
CD46 is expressed on HSPCs permitting transduction with an Ad5/F35-based adenovirus vector expressing GFP.
PMA, bryostatin dose response, and adenoviral vector MOI on CCR5 disruption and viability of HSPC CD34+
Viability of PMA-treated HSPC following Ad5/F35 CCR5-ZFN or Ad5/F35 hIL2Rg10 transduction.
CCR5-ZFN–induced CCR5 disruption is sustained and present in multiple lineages.
Multiple lineage engraftment of PKC activator-treated and CCR5-ZFN–transduced HSPCs in the spleen and bone marrow of transplanted NSG mice.
CCR5 disruption was present and enhanced in multiple lineages in long-term transplanted, HIV-1–challenged NSG mice.
Off-site target analysis of PMA-treated and Ad/35ZFN-transduced cells.
List of genes in which off-target cleavage may occur.