Our major goal in these studies is to introduce both CXCR4 and CCR5 siRNAs into a single lentiviral construct to achieve their stable expression in transduced cells. Lentiviral vectors offer advantages over conventional retroviral vector systems since they can transduce dividing as well as nondividing cells and are less prone to transgene silencing 44454647. The transfer vector HIV-7-GFP-XHR (referred to as XHR) contained a short hairpin type anti-CXCR4 siRNA driven by a Pol-III U6 promoter followed by a short hairpin anti-CCR5 siRNA driven by a different Pol-III promoter, H1. Downstream, the reporter gene, EGFP is driven by a CMV promoter. The control GFP-alone vector, HIV-7-GFP, contained only the reporter gene EGFP (Fig 1).

Magi-CXCR4 cells constitutively expressing CXCR4 on the cell surface when transduced with the control vector or XHR vector had shown 97% and 83% EGFP expression respectively as measured by FACS analysis indicating high efficiency of transduction (Fig 2A and 2C). To determine if CXCR4 was down regulated by the respective siRNA in the XHR construct, the transduced cells were analyzed for CXCR4 surface expression. The surface levels of CXCR4 were reduced significantly in XHR transduced cells (73% lower) compared to the cells transduced with control vector (Fig 2B and 2D) indicating the efficacy of the CXCR4 siRNA on its target. Similarly, to determine the activity of the anti-CCR5 siRNA in the XHR vector, transduced Ghost R5 cells that constitutively express CCR5 were evaluated. As seen in Fig 3A and 3C, high levels of transduction (84% and 83%) were seen in Ghost-R5 cells with either the control vector or XHR vector, respectively. When the transduced cells were analyzed for CCR5 expression, a dramatic decrease in CCR5 expression was seen in XHR cells (72%) compared to control vector transduced cells (Fig 3B and 3D). These results had shown that the bispecific lentiviral vector XHR efficiently down regulates both CXCR4 and CCR5 targets in respective cells.

To confirm that the down regulation of both CXCR4 and CCR5 coreceptors as seen by FACS analysis is due to reduced levels of the corresponding mRNAs, vector transduced cells were analyzed by RT-PCR. As an internal control, GAPDH mRNA was also analyzed. XHR vector transduced cells showed considerable reduction in transcript levels for both CXCR4 and CCR5 as compared to non-transduced and control GFP vector transduced cells. The levels of GAPDH control mRNA remained unchanged in all samples (Fig 4). To validate the expression of individual siRNAs in transduced Magi-CXCR4 and Ghost R5 cells, cellular RNA was analyzed by northern analysis for their presence. As internal controls, the presence of constitutively expressed miRNA-16 RNAs were also analyzed in parallel. As expected, comparable levels of miRNA-16 RNAs (22 bp in length)were detected in GFP control vector transduced as well as in XHR vector transduced cells (Fig 5A). RNAs corresponding to CXCR4 and CCR5 shRNAs (representing the 21nt antisense strand of each shRNA) were seen in XHR transduced but not in GFP control vector transduced cells (Fig 5B).

Double stranded RNA molecules longer than ~30 bp are known to induce the interferon pathway in response to viral infections. As siRNAs are generally comprised of 19–24 bp in length, they are not expected to activate such a response that mediates a non-specific down regulation of cellular or viral mRNAs. However, recent data had shown that in some circumstances, certain siRNAs might induce variable levels of interferon activation 484950. To rule out such a possibility with the present siRNAs, we looked for upregulation of phosphorylated-PKR by western blot analysis. PKR is a protein kinase that becomes activated through phosphorylation in the presence of dsRNA and is involved during the interferon response. Our results have shown that the levels of phosphorylated PKR remain unchanged in XHR transduced cells similar to mock and GFP vector transduced cells. In contrast, elevated levels of phosphorylated PKR could be seen in poly I:C transfected cells used as positive controls (Fig 6). These data exclude the possibility of non-specific interferon activation by the combinatorial lentiviral construct.

To determine if down regulation of the essential coreceptors, CXCR4 and CCR5, translated to virus resistance, transduced Magi-CXCR4 and Ghost R5 cells were challenged with X4 (NL4-3) and R5 (BaL1)-tropic strains of HIV-1 respectively. Viral p24 antigen levels at different days post-challenge were determined by ELISA to quantify levels of HIV-1 resistance. Over a 10-fold reduction in viral antigen levels was seen with both XHR transduced Magi-CXCR4 and Ghost-R5 cells as compared to non-transduced and GFP-alone vector transduced cells (Fig 7). There was a slight increase in viral production in XHR transduced cells on days 5 to 7. This could be due to nontransduced and/or low siRNA expressing cells producing the virus. We next wanted to determine if the XHR vector expressing CXCR4 and CCR5 siRNAs is effective in physiologically relevant cells for gene therapy. Accordingly, PBMCs transduced with vectors were challenged in the same manner as above. A 3-fold level of inhibition was seen on days 3, 5, and 7 (Fig 8). These results established that the XHR vector is also effective in primary cells in inhibiting HIV-1. Although clearly significant, the levels of virus inhibition were not as dramatic as seen with Magi and Ghost cell lines. The observed levels of viral inhibition in primary PBMC are similar to those observed in a recent report 31. Lower levels of protection in PBMCs were likely due to the lower levels of transduction. Future studies that are aimed at increasing transduction efficiencies into primary lymphocytes and macrophages are likely to overcome this hurdle.

In summary, our studies have shown for the first time that a single lentiviral vector could be used to stably deliver two different siRNAs targeted to two different cell surface co-receptor molecules and achieve protection against both X4 and R5 tropic HIV-1 viral strains. The short hairpin design permitted use of a single promoter to transcribe both the sense and anti-sense strands of each of the siRNAs. No promoter interference was observed between the U6 promoter driving the transcription of CXCR4 siRNA and the H1 promoter driving the CCR5 siRNA since comparable amounts of both the siRNAs could be seen in transduced cells. Furthermore, possible interferon induction by the combinatorial construct was also ruled out.

A major advantage in using a combinatorial lentiviral construct targeted to both the coreceptors is that infection with either of the viral strains could be prevented at the entry step thus eliminating the possibility of proviral integration and viral latency. Given the success with the current bispecific construct, other novel constructs could be designed and experimented with that incorporate siRNAs targeted to both the cellular as well as viral targets. Based on the design employed here, it is possible to introduce more than two siRNAs in a single construct in the future. However caution should be exercised while incorporating multiple siRNAs in a single construct because the possibility exists that over expression of foreign siRNAs in a cell may have undesirable effects such as saturating the endogenous RISC complex and consequent toxicity. Such a possibility needs to be tested in long range experiments in vivo. We previously have introduced a monospecific siRNA targeted to HIV-1 rev into CD34 hematopoietic progenitor cells via lentiviral vectors and derived transgenic macrophages in vitro and T cells in vivo 29. The transgenic cells were found to be apparently normal while markedly resistant to HIV-1 infection.

No deleterious effects are expected by the stable knock down of the CCR5 coreceptor in vivo since individuals harboring a 32 bp deletion in the corresponding gene are physiologically normal 3435. Although CXCR4 down regulation in circulating mature T cells in the periphery may not have any insurmountable ill effects, this may have possible drawbacks in a stem cell setting due to its role in cell homing into bone marrow 5152. Additionally, recent gene expression profiling studies indicated some off-target effects by siRNAs 53. Therefore, the present combinatorial construct targeted to both CXCR4 and CCR5 coreceptor molecules need to be thoroughly tested in an in vivo system such as the SCID-hu mouse model to evaluate its efficacy and possible toxicity in differentiated cells before it can be used for gene therapy in human subjects. Such experiments are currently underway.

Previously characterized siRNAs against CXCR4 and CCR5 were used in generating the bispecific lentiviral vector 232430. A third generation lentiviral vector backbone was employed to derive the bispecific constructs. The two cis-acting elements, namely, the central DNA flap consisting of cPPT and CTS (to facilitate the nuclear import of the viral preintegration complex) and the WPRE (to promote nuclear export of transcripts and/or increase the efficiency of polyadenylation of transcripts), are engineered to enhance the performance of the vector 3839. An siRNA expression cassette targeting CXCR4 under the control of the Pol-III U6 promoter was PCR amplified from the plasmid pTZ-U6+1 as described by Castanotto et al 40. This cassette was cloned into pHIV-7-GFP transfer vector in the BamHI site immediately upstream of the CMV-EGFP gene. This cassette contained a MluI restriction site downstream from the CXCR4 siRNA sequence for subsequent cloning of the H1 promoter driven CCR5 siRNA cassette. The H1-CCR5 siRNA expression cassette was also generated as described above using the plasmid pSUPER (Oligoengine, Seattle, WA). Sequencing and confirmation of candidate clones was performed by Laragen Inc. (Los Angeles, CA). The transfer vector containing the inserts U6-X4 siRNA and H1-CCR5 siRNA is termed pHIV-XHR-GFP.

293T cells and PBMCs were maintained in DMEM media supplemented with 10% FBS. Magi-CXCR4 cells obtained from the AIDS Reference and Reagent Program were maintained in media as previously described 4142. Ghost-R5 cells obtained from the AIDS Reference and Reagent Program were maintained in media as previously described 43. To generate lentiviral vectors, fifteen micrograms of transfer vector with either GFP-alone or XHR were transfected along with 15 ug pCHGP-2, 5 ug pCMV-Rev, and 5 ug pCMV-VSVG into 293T cells at 60% confluency in 100 mm culture dishes using a calcium phosphate transfection kit (Sigma-Aldrich, St. Louis, MO). Six hours after transfection, fresh medium was exchanged. Cell culture supernatants containing the vector were collected at 24, 36, 48, and 60 hours post transfection and pooled. Vector supernatants were concentrated by ultracentrifugation and later titrated on 293T cells using FACS analysis for GFP expression.

Magi-CXCR4 and Ghost-CCR5 cells were seeded in 6-well plates 24 hours prior to transduction, 5 × 10⁵ cells per well. Cells were transduced with lentiviral vectors at an m.o.i. of 10 in the presence of 4 ug/ml polybrene for 2 hours. For transduction of PBMCs, cells were first isolated from whole blood by Histopaque^®-1077 (Sigma-Aldrich), and then cultured in CD3 and CD28 antibody coated plates. Three days after stimulation, PBMCs were transduced at an m.o.i of 20 in the presence of 4 ug/ml polybrene. PBMC transduction was repeated the following day. Seventy-two hours post transduction with siRNA containing lentiviral vectors, FACS analysis was performed to determine the levels of cell surface expression of CXCR4 and CCR5. Non-transduced and transduced cells were stained with appropriate antibodies conjugated with PE-Cy 5 (Pharmingen, San Diego, CA) namely, anti-CXCR4 for Magi-CXCR4 cells and anti-CCR5 for Ghost-CCR5 cells. Transduction efficiency was determined by assaying for EGFP expression. FACS analysis was performed on the Beckman Coulter Epics XL using ADC software for analysis.

Total RNA was extracted from non-transduced and transduced Magi-CXCR4 and Ghost-CCR5 cells using the RNA-STAT-60 reagent (Tel-Test, Friendswood, TX). Small RNAs, <200 nt, were separated and concentrated using the mirVana™ miRNA Isolation Kit (Ambion, Austin, TX). Twenty micrograms of small RNAs were hybridized overnight at 37°C using the mirVana™ miRNA Detection Kit (Ambion) with γ-³²P labeled probes made using the mirVana™ Probe & Marker Kit (Ambion). Probes were complementary to the antisense strands of CXCR4 and CCR5 siRNAs. Hybridization reactions were processed according to the manufacturer's protocol and run on 15% polyacrylamide TBE-Urea gels. Gels were then exposed to X-ray film. A probe complementary to miRNA-16 supplied with the miRNA detection kit was used as an internal control.

Cell lysates of non-transduced and transduced cells were run on 10%-polyacrylamide-SDS TBE gels. Proteins were immunoblotted onto Immobilon™-P membranes (Millipore, Bedford, MA) and incubated with antibody specific for phosphorylated-PKR (Sigma-Aldrich), while anti-actin antibody (Sigma-Aldrich) was used to detect cellular actin as an internal control. A secondary antibody, goat anti-rabbit IgG conjugated with alkaline phophatase (Promega, Madison, WI), was then added. An alkaline phophatase substrate reagent, Western Blue (Promega), was used to visualize the bands.

Total RNA was extracted from non-transduced and transduced cells. Primers specific for CXCR4 (forward: 5'-ggaggggatcagtatatacacttc and reverse: 5'-cgccaacatagaccaccttttc) and CCR5 (forward: 5'-caaaaagaaggtcttcattacacc and reverse: 5'-cttgctcgctcgggagcctc) (IDT, Coralsville, IA) were used to determine transcript levels while GAPDH (forward: 5'-ctgagaacgggaagcttgtcatcaa and reverse: 5'-gcctgcttcaccaccttcttgatg) primers were used as an internal control. One-step RT-PCR reactions were performed using the Superscript™ III One-Step RT-PCR kit (Invitrogen, Carlsbad, CA). Reactions were run on 1% agarose gels and appropriate bands were visualized with UV light.

To determine if down-regulation of CXCR4 and CCR5 transcript levels and cell surface expression inhibited HIV-1 infection, non-transduced and transduced cells were challenged with NL4-3 (X4-tropic) and BaL-1 (R5-tropic) strains of HIV-1, at an m.o.i of 0.01, as previously described 24. Viral supernatants were collected daily from infected Magi-CXCR4 and Ghost-CCR5 cells for p24 assay. ELISA was used to determine p24 values employing a Coulter-p24 kit (Beckman Coulter, Fullerton, CA). For PBMC challenge experiments, non-transduced and transduced cells were infected with NL4-3 and Bal-1 strains and cell culture supernatants were collected on days 1, 3, 5, and 7 post-infection to measure p24 levels.