Materials. Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St Louis, MO), all restriction enzymes were obtained from New England BioLabs (Ipswich, MA) and all cell culture products were purchased from GIBCO (Gibco BRL/Life Technologies, a division of Invitrogen, Grand Island, NY). Random primers (Invitrogen, Carlsbad, CA); Lipofectamine 2000 (Invitrogen); Random primers (Invitrogen); HEK 293 and CCRF-CEM (ATCC); CHO-Env Transfectants (CHO-WT and CHO-EE); the HIV-1 NL4.3 and HIV-1 IIIB viruses were obtained from the AIDS Research and Reference Reagent Program.
siRNAs. DsiRNAs were purchased from Integrated DNA Technologies (Coralville, IA).
Anti-tat/rev Site I 27-mer: Sense: 5′-GCG GAG ACA GCG ACG AAG AGC UCA UCA-3′ antisense: 5′-UGA UGA GCU CUU CGU CGC UGU CUC CGC dTdT-3′.
Anti-CD4 27-mer: Sense: 5′-GAU CAA GAG ACU CCU CAG UGA GAA G-3′ antisense: 5′-CUU CUC ACU GAG GAG UCU CUU GAU CUG-3′ (2′-OMe modified U was underlined.)
Anti-TNPO3 27-mer: Sense: 5′-CGA CAU UGC AGC UCG UGU ACC AG dGdC-3′ antisense: 5′-GCC UGG UAC ACG AGC UGC AAU GUC GUU-3′.
A-1-stick, stick-sense, and stick-antisense were chemically synthesized by the Synthetic and Biopolymer Chemistry Core in the City of Hope. And the corresponding stick-siRNAs and aptamer-stick-siRNAs were formed as previously described.45
A-1-stick: 5′-GGGAGGACGAUGCGGAAUUGAGGGACCACGCGCUGCUUGUUGUGAUAAGCAGUUUGUCGUGAUGGCAGACGACUCGCCCGA XXXXXXXGUACAUUCUAGAUAGCC-3′
Sense strand: 5′-GCGGAGACAGCGACGAAGAGCUCAUCAUU-3′
Antisense strand: 5′-UGAUGAGCUCUUCGUCGCUGUCUCCGC UdT-3′
Antisense-stick: 5′-UACACGAGCUGCAAUGUCGGCUUUGCU XXXXXGGCUAUCUAGAAUGUAC-3′
Sense strand: 5′-CAAAGCCGACAUUGCAGCUCGUGUAUU-3′
Antisense strand: 5′-GCCUGGUACACGAGCUGCAAUGUCG UU-3′
Stick-sense: 5′-UCAAGAGACUCCUCAGUGAGAAGAA XXXXXGGCUAUCUAGAAUGUAC-3′
Antisense strand: 5′-UUCUUCUCACUGAGGAGUCUCUUGAUU-3′
The underlined nucleotides corresponds to the stick sequence. The stick sequence contains 2′-OMe–modified A and G and 2′-F–modified U and C. The 2′-F–modified U and C are highlighted as bolded text. The italic X indicates the 3 carbon linker (C3) between the aptamer/siRNA and stick sequences.
Generation and HIV-1 infection of humanized Rag2−/−γc−/− mice (RAG-hu mice).
mice were prepared as previously described using human fetal liver-derived CD34+ cells.28,46
Briefly, neonatal mice were conditioned by irradiating at 350 rads and then injected intrahepatically with 0.5–1 × 106
human CD34+ cells. Approximately 12 weeks post-reconstitution, mice were screened for human cell engraftment. Blood was collected by tail bleeds, and RBCs were lysed using the Whole Blood Erythrocyte Lysing Kit (R&D Systems, Minneapolis, MN). The white blood cell fraction was stained with antibodies against the human pan-leukocyte marker CD45 (Caltag, a division of Invitrogen, Grand Island, NY) and FACS analyzed as described. To infect human cell reconstituted RAG-hu mice, HIV-1 NL4-3 (1.2 × 105
viral load IU/ml) in a 100 µl volume was injected intraperitoneally at least 12 weeks after cell engraftment. Viral loads were examined weekly and viremia was established in all the mice by 4 weeks. Treatment was done by intravenous injection on the last day of week 4 with 0.25 nmol experimental RNAs (4.6 µg cocktailed DsiRNAs including equal amount of three DsiRNAs: tat/rev
DsiRNA, TNPO3 DsiRNA, and CD4 DsiRNA, or 14.1 µg A-1-stick–cocktailed DsiRNAs conjugates with equal amount of three DsiRNA portions) in a 40 µl volume, followed by another the next day. Later, the injections were continued on a weekly basis for 4 weeks. In the second in vivo
treatment experiment, 0.25 nmol conjugates in a 40 µl volume were administered at 23 and 24 weeks of post-infection like above.
Measurement of viral load in plasma. To quantify cell-free HIV-1 by qRT-PCR, RNA was extracted from 25 to 50 µl of EDTA-treated plasma using the QIAamp Viral RNA kit (QIAGEN, Valencia, CA). cDNAs were produced with Superscript III reverse transcriptase (Invitrogen) using a primer set specific for the HIV-1 long terminal repeat sequence, and qPCR was performed with the same primer set and a long terminal repeat-specific probe using Supermix UDG (Invitrogen) as described.
Flow cytometry. Whole blood was collected and RBCs were lysed as reported previously. Peripheral blood cells were stained for hCD3-PE and hCD4-PECy5 (Caltag) markers and analyzed using a Coulter EPICS XL-MCL FACS analyzer (Beckman Coulter, Brea, CA). CD4+ T-cell levels were calculated as a ratio of the entire CD3 population (CD4 + CD3+:CD4 − CD3+). To establish baseline CD4+ T-cell ratios, all mice were analyzed before infection.
Detection of tat/rev siRNA. At 1, 3, and 9 weeks post-injection, blood samples were collected and small RNAs were isolated with MirVana miRNA isolation Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instruction. The siRNA quantification was performed using TaqMan MicroRNA Assay according to manufacturer's recommended protocol (Applied Biosystems). Ten nanogram of small RNA, 0.2 µmol/l stem-loop RT primer, RT buffer, 0.25 mmol/l dNTPs, 3.33 units/ml MultiScribe RT, and 0.25 units/ml RNase inhibitor were used in 15 µl RT reactions for 30 minutes at 16 °C, 30 minutes at 42 °C, and 5 minutes at 85 °C, using the TaqMan MicroRNA reverse transcription Kit (Applied Biosystems). For real-time PCR, 1.33 µl of cDNA, 0.2 mmol/l TaqMan Probe, 1.5 mmol/l forward primer, 0.7 mmol/l reverse primer, and TaqMan Universal PCR Master Mix were added in 20 µl reactions for 10 minutes at 95 °C and 40 cycles of 15 seconds at 95 °C and 1 minute at 60 °C. All real-time PCR experiments were done using an iCycler iQ system (Bio-Rad, Hercules, CA). Primers were as follows: Site I Looped RT primer: 5′-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA CAG CG-3′ Site I forward primer: 5′-GCT GAT GAG CTC TTC GTC G-3′ Site I reverse primer: 5′-GTG CAG GGT CCG AGG T-3′ Site I probe primer: 5′-6- FAM- TCG CAC TGG ATA CGA CAC AGC GAC GA –BHQ1-3′. In this case, a synthetic 27-mer duplex RNA was used as positive control.
Assays of targeted gene expression. Human PBMCs were obtained from transplanted mice at 1 and 3 weeks post-injection and total RNAs were isolated with STAT-60 (TEL-TEST “B”) according to the manufacturer's instructions. Residual DNA was digested using the DNA-free kit per the manufacturer's instructions (Ambion, a division of Invitrogen, Grand Island, NY). cDNA was made using 2 µg of total RNA. RT was carried out using Moloney murine leukemia virus reverse transcriptase and random primers in a 15 µl reaction according to the manufacturer's instructions (Invitrogen). Expression of the tat/rev coding RNAs was analyzed by qRT-PCR using 2× iQ SyberGreen Mastermix (BIO-RAD) and specific primer sets at a final concentration of 400 nmol/l. Gapdh expression was used for normalization of the qPCR data. Primers were as follows: IIIB or NL4-3 tat/rev forward primer: 5′-GGC GTT ACT CGA CAG AGG AG -3′ IIIB or NL4-3 tat/rev reverse primer: 5′-TGC TTT GAT AGA GAA GCT TGA TG-3′ CD4 forward primer: 5′-GCT GGA ATC CAA CAT CAA GG-3′ CD4 reverse primer: 5′-CTT CTG AAA CCG GTG AGG AC-3′ TNPO3 forward primer: 5′-CCT GGA AGG GAT GTG TGC-3′ TNPO3 reverse primer: 5′-AAA AAG GCA AAG AAG TCA CAT CA-3′ gapdh forward primer 1: 5′-CAT TGA CCT CAA CTA CAT G-3′ gapdh reverse primer 2: 5′-TCT CCA TGG TGG TGA AGA C-3′.
Interferon assays. Total RNA was isolated from PBMCs of treated mice using STAT-60. Expression of mRNAs encoding p56 (CDKL2) and OAS1 were analyzed by qRT-PCR using 2× iQ SyberGreen Mastermix (BIO-RAD) as described above and specific primer sets for these genes at final concentrations of 400 nmol/l. Primers were as follows: P56 (CDKL2) forward, 5′-TCA AGT ATG GCA AGG CTG TG-3′ P56 (CDKL2) reverse, 5′-GAG GCT CTG CTT CTG CAT CT-3′ OAS1 forward, 5′-ACC GTC TTG GAA CTG GTC AC-3′ OAS1 reverse, 5′-ATG TTC CTT GTT GGG TCA GC-3′ gapdh expression was used for normalization of the qPCR data.
To measure any induced IFN-α directly, human IFN-α1 ELISA Ready-SET-Go! (eBioscience, San Diego, CA) was used. Mice were injected with the naked cocktailed DsiRNA or ptamer-cocktailed DsiRNA conjugates. At 2 and 24 hours post-treatment, 25–50 µl of EDTA-treated plasma was collected from three mice per treatment group and from three positive control mice which had been intravenously injected with 5 µg of poly (I:C) (Sigma-Aldrich) in a 50 µl volume. Plasma levels were evaluated as per instructions supplied in the kit.
The mouse viral loads and CD4:CD3 T-cell ratios were plotted by using a Lowess smoother across values. Viral loads were first log-transformed before smoothing and then anti-transformed for plotting. Missing values were imputed with a last observation carried forward scheme. The calculations were conducted as previously described.25,47
HIV-1 suppression by chemically synthesized aptamer (A-1-stick) and aptamer-stick-siRNA conjugates.
Chemically synthesized aptamer-stick-siRNA conjugates are processed by Dicer and induce mRNA cleavage.
2′-Fluoro–modified aptamer-stick-siRNA conjugate in mouse serum.
HIV-1 viral loads in individual RAG-hu mice.
CD4 T-cell levels in individual treated and control RAG-hu mice.
Sequence analysis of mutant virus from animals treated with aptamer-stick–cocktailed DsiRNA conjugates.
Materials and Methods.