Background
HIV/AIDS continues to be a major public health problem worldwide with millions of people currently infected and new infections being on the rise. As no effective vaccines are currently available for prevention, new and innovative therapies need to be developed. Although combinatorial therapies such as HAART have proven to be effective in prolonging life, they do not afford a complete cure. Other constraints with HAART therapy are the development of drug resistant viral mutants and toxicity after prolonged therapy. Intracellular immunization by gene therapy strategies offers a promising alternative approach for controlling and managing HIV disease. A number of previous approaches that involved the use of transdominant proteins [
1‐
3], decoys [
3‐
7], and ribozymes [
5,
8‐
12] had shown initial promise but fell short of practical utility in providing adequate protection. With the discovery that the RNA interference phenomenon operates in mammalian cells and is highly effective in selective gene silencing, new potent small interfering RNA (siRNA) molecules have become available to add to the anti-HIV arsenal [
13].
RNAi is a highly potent mechanism of post-transcriptional gene silencing. Mediated by sequence specific siRNAs, it can effectively down regulate expression of either viral or cellular RNA target molecules by selective degradation of mRNAs [
13‐
16]. Mechanism of destruction involves an endonuclease present in the RISC complex which is guided by the antisense component of the siRNA for target recognition. A number of reports have shown that delivery of siRNAs by transfection of presynthesized or plasmids encoding siRNAs into cultured cells can effectively inhibit HIV-1 infections [
17‐
26]. Antiviral effects of these delivery methods are only transient due to eventual degradation and dilution of siRNAs during cell division. For HIV gene therapy strategies to succeed in long range, it is necessary that siRNA coding transgenes be maintained and expressed long term in a virus susceptible target cell. In this regard, lentiviral vectors have proven to be highly effective in high efficiency gene transduction and sustained gene expression.
A number of previous approaches using either synthetic siRNAs or plasmid expressed constructs have successfully targeted viral transcripts and achieved effective viral inhibition. Of these, some anti-HIV-1 siRNAs, such as siRNAs against tat, tat-rev had been introduced into lentiviral vectors and their efficacy was demonstrated both in cell lines and primary T cells and macrophages [
27,
28]. Promising data was also obtained in experiments showing that anti-rev siRNAs against HIV-1 were functional in conferring viral resistance in differentiated T cells and macrophages derived from lentiviral transduced CD34+ hematopoietic progenitor cells [
29].
In addition to targeting viral transcripts, many studies including ours also investigated the efficacy of siRNAs in down regulating host cell molecules necessary for HIV-1 infection [
18,
21,
23,
24,
30,
31]. An advantage in targeting cellular molecules is that efficacy will be more broad spectrum against all the clades of the virus and the frequency of escape mutants will be lower. Down regulation of the primary cell surface receptor CD4 and consequent inhibition of HIV-1 infection was shown using synthetic siRNAs. However, since CD4 is an essential cell surface molecule for immunological function, it is not a practical target for HIV gene therapy. Chemokine receptors CCR5 and CXCR4 play critical roles as coreceptors for viral entry during infection with macrophage tropic R5 and T cell tropic X4 HIV-1 viral strains respectively [
32,
33]. Thus they are suitable targets for siRNA mediated down regulation. Since both R5 and X4 viral strains are involved in disease pathogenesis, it is important to consider blocking of both respective coreceptors when developing effective therapeutics. In a segment of the human population, a naturally occurring 32-bp deletion in the CCR5 gene results in the loss of this coreceptor thus conferring significant resistance to HIV infection [
34‐
36]. Homozygous or heterozygous individuals for this mutation remain physiologically normal. With regard to the CXCR4 coreceptor, it was found to be dispensable for T cell development and maturation in murine studies [
37]. These findings suggest that CCR5 and CXCR4 are promising targets for HIV therapies.
Based on this rationale, recent work with synthetic siRNAs demonstrated that down regulating either CXCR4 or CCR5 will protect cells from X4 or R5 HIV-1 strains respectively at the level of viral entry [
18,
21,
23,
24]. Although stable expression of an anti-CCR5 siRNA was achieved using a lentiviral vector in one study, down regulating CCR5 alone in the face of an HIV-1 infection is insufficient [
31]. Therefore, we recently experimented with synthetic bispecific combinatorial constructs targeted to both CXCR4 and CCR5 and have shown their efficacy in cultured cells [
24]. To make further progress, our present studies are directed towards constructing a single bispecific lentiviral vector expressing both CXCR4 and CCR5 siRNAs. Using this combinatorial construct, here we show high efficiency transduction, simultaneous down regulation of both coreceptors resulting in HIV-1 resistance.
Conclusions
For HIV/AIDS gene therapy strategies to succeed, novel molecules need to be harnessed. In this regard, siRNAs offer great potential. Exploitation of these promising candidates to down regulate essential cellular coreceptors via the use of lentiviral vectors facilitates long term derivation of resistant T cells and macrophages which are the main targets for the virus. Our results showed for the first time that expression of both CXCR4 and CCR5 siRNAs in combination is possible by the use of lentiviral vectors. Coreceptor specific siRNAs stably transduced with the bispecific lentiviral vector showed marked resistance against both T cell tropic and monocyte tropic HIV-1 infection in cell lines and primary PBMCs. The newly developed bispecific vector shows promise for potential in vivo application.
Materials and Methods
Plasmid and lentiviral vector construction
Previously characterized siRNAs against CXCR4 and CCR5 were used in generating the bispecific lentiviral vector [
23,
24,
30]. 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 [
38,
39]. 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
BamH I site immediately upstream of the CMV-EGFP gene. This cassette contained a
Mlu I 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.
Cell culture and vector production
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 [
41,
42]. 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.
Lentiviral vector transduction and FACS analysis
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.
Northern analysis for shRNA expression
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 mir Vana™ miRNA Isolation Kit (Ambion, Austin, TX). Twenty micrograms of small RNAs were hybridized overnight at 37°C using the mir Vana™ miRNA Detection Kit (Ambion) with γ-32P labeled probes made using the mir Vana™ 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.
Western Blot analysis of phosphorylated PKR
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.
RT-PCR
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.
HIV-1 Challenge
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.
Competing interests
The author(s) declare that they have no competing interests.
Author's contributions
JA carried out all of the experiments. RA was responsible for the overall experimental design and implementation of the project.