Tar decoy and Tar-CCR5Rz containing lentiviral vectors
The design, structure, and in vitro efficacy in cultured cells of anti-CCR5 ribozyme, Tar decoy, and Tar-CCR5Rz constructs were described previously [5, 10, 18, 19]. These inhibitory RNAs introduced into a third generation self-inactivating lentiviral vector were used in the present study . The transfer vector pHIV-7-GFP containing a CMV driven EGFP reporter gene is depicted in Fig. 1, panel 1A. Two important unique features are the cis-acting elements, the HIV-1 central flap sequence and the woodchuck post-transcriptional regulatory element (WPRE) for optimal EGFP expression. In the transfer vector pHIV-U16-Tar-GFP, the Tar decoy under the control of the U6 promoter was positioned upstream of the EGFP reporter (Fig 1B). In the combinatorial construct pHIV-U16Tar-CCR5Rz-GFP, the Tar decoy is driven by U6 whereas the CCR5 ribozyme is under the control of the VA1 promoter (Fig. 1C).
Production of high titered retroviral vectors
To generate vector stocks, 293T cells were transfected with 15 μg of pCHGP-2 (encodes HIV-1 gag/pol), 15 μg of transfer vector (pHIV-7 GFP or pHIV-U16 Tar-GFP or pHIV-U16Tar-CCR5Rz-GFP), 5 μg of pCMV-rev and pCMV-G each as described previously . Viral supernatants were collected at 24, 48 and, 72 hrs post transfection, pooled, and concentrated by ultracentrifugation . Concentrated virus was resuspended in a small volume (500 μl) of DMEM containing 10% fetal bovine serum. The titer of the vector preparation was determined in 293T cells as described previously and ranged from 1 to 3 × 108 TU/ml. Multiple aliquots were made and stored at -70°C.
Isolation of CD34+ hematopoietic progenitor cells and high efficiency vector transduction
Human fetal liver CD34+ hematopoietic progenitor cells (HPC) were purified by positive selection on a magnetic column using the Direct CD34 Progenitor Cell Isolation Kit from Miltenyi Biotech, Gladbach, Germany, as described in detail earlier . Purified cells were suspended in Iscove's medium supplemented with IL3, IL6, and human stem cell factor (SCF), each at a concentration of 100 ng/ml (R & D Systems, Minneapolis, MN) and cultured for 15 hours at 37°C. Vector transductions were carried out in a 12-well tissue culture plate using 2 × 106 cells at an m.o.i. of 10 to 20 in a final volume of 100 μl of medium containing 4 μg/ml polybrene. Following transduction, cell aliquots were used for carrying out in vitro colony forming unit (CFU) assays, generation of macrophages, and for reconstitution of human thy/liv grafts in SCID-hu mice to generate T cells.
CFU assays and generation of macrophages
Control, or vector transduced CD34+ cells were allowed to differentiate into multiple lineages of erythroid and myeloid lineages in a semi-solid medium (MethocultTM GF H4434, Stem cell Technologies, Vancouver, BC, Canada). This medium contains the following components: 1% Methylcellulose in Iscove's MDM, 30% fetal bovine serum, 1% bovine serum albumnin, 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 50 ng/ml rh stem cell factor, 10 ng/ml rh GM-CSF, 10 ng/ml rh IL-3, and 3 units/ml rh erythropoietin. A colony forming unit (CFU) was defined as having at least 50 cells after 14 days in the above selective medium. Individual myelomonocytic colonies were pooled and cultured in DMEM supplemented with 10% fetal bovine serum, 50 ng/ml M-CSF, and 20 ng/ml GM-CSF, for a period of 14 days for differentiation into macrophages. Cells were stained for CD14 antigen and analyzed by FACS to determine macrophage yield.
Reconstitution of SCID-hu grafts with transduced CD34+ cells and derivation of T cells
Human fetal thymus and liver tissues were implanted under the kidney capsule of SCID mice to generate SCID-hu mice as described earlier . Control and lentivirus vector transduced CD34+ progenitor cells (1 × 106) were injected directly into thy/liv grafts for reconstitution. Eight to ten weeks post reconstitution, thus allowing for T cell differentiation, the animals were sacrificed and thymocytes were isolated from the grafts. The differentiated thymocytes were cultured in vitro and checked for their ability to respond to mitogen, PHA-P and interleukin-2. Briefly, thymocytes were washed once with medium containing serum and resuspended into Iscove's medium supplemented with 10% fetal bovine serum. Approximately 2 × 106 cells were plated in a 12 well tissue culture plate and stimulated with PHA-P (4 μg/ml) and IL-2 (10 U/ml) for three days. Cells were counted after 3 days to determine expansion.
PCR Detection of Tar RNA
Total RNA was isolated from approximately 1 × 106 control and transgenic macrophages using Qiagen RNA/DNA mini kit (Qiagen, Germany) and subjected to RT-PCR as described before . In each case, a 125 bp DNA fragment is expected. The following primers were used: 1, Forward Tar: 5'-GCAATGATGTCGTAATTTGC and 2, Reverse Tar: 5'-CTTGCTCAGTAAGAATTTTCGTC.
HIV-1 infection of thymocytes
Thymocytes derived from thy/liv grafts of SCID-hu mice were sorted by FACS to enrich for EGFP expressing cells (>90% purity). They were later expanded by stimulation by PHA-P in medium containing serum and IL-2 as described earlier [6, 23]. Approximately 106 cells were infected with HIV-1 NL4-3 at an m.o.i. of 0.001 in a final volume of 100 μl for 3 hrs at 37°C. Infected cells were washed twice with DMEM with 10% fetal bovine serum and cultured in a 12 well plate for 3 weeks. Supernatants (0.5 ml) were collected on alternative days with media replenished in each well. Amounts of virus produced in cell culture supernatants was measured by HIV-1 p24 ELISA.
HIV-1 challenge of CD34+ cell derived macrophages
Infection with a macrophage-tropic Bal-1 strain of HIV-1 was carried out in a 6 well plate. Approximately 2 × 106 adherent macrophages differentiated in vitro were infected with Bal-1 virus at an m.o.i. of 0.001 in the presence of 4 μg/ml of polybrene for 6 hours. Thereafter, 3 ml of DMEM supplemented with 10% serum was added. Supernatants (0.5 ml) were collected every other day from each well for 3 weeks and stored at -70°C. The levels of virus released were determined by p24 antigen ELISA.