Introduction

RNA interference (RNAi) refers to posttranslational and posttranscriptional gene silencing mediated by single- and double-stranded RNA. In mammalian cell culture, RNAi can be engineered through either liposomal transfection of preformed short interfering RNA (siRNA) or vectors encoding siRNA expression cassettes^1,2,3,4. A challenge is to harness the specificity and potency of RNAi for therapeutic gain in humans. Using animal models, several investigators have demonstrated that the direct tissue or intravascular injection of synthetic siRNA or vectors expressing siRNA or short hairpin RNA (shRNA) leads to the desired gene silencing in targeted tissues^5,6,7,8. Recently, the mucosal application of siRNA targeting viral factors led to protection in mice challenged with respiratory and sexually transmitted viruses (e.g., syncytial and parainfluenza virus, herpes simplex virus-2)^9,10,11. We reasoned that host gene expression in vaginal and colonic mucosa could serve as another target of siRNA activity with therapeutic promise. Here, we examine in detail the gene silencing durability and specificity of mucosal RNAi and its therapeutic potential in an animal model of inflammatory bowel disease.

Results and discussion

On a simplistic level, the mucosa can be deconstructed into its somatic and hematopoietic cellular compartments. We chose constitutively expressed proteins in both compartments as siRNA targets and determined the kinetics of gene silencing in vivo. As a somatic target, we chose the nuclear membrane protein lamin A/C. We used quantitative real-time RT-PCR to confirm high levels of lamin A/C mRNA expression (2.6 10⁶ copies of mRNA/g total RNA input) in the cervicovaginal tract of C57BL/6 mice. Given previous demonstrations of mucosal uptake of oligonucleotides after direct application, we administered fixed doses (20, 40, 60 nmol) of lamin A/C siRNA in a variety of resuspension solutions to the murine vagina^12,13. Each treatment group consisted of four or five animals. Forty-eight hours later, we euthanized the animals and dissected and processed vaginal tissue for mRNA extraction. The application of naked siRNA did not lead to a statistically significant decrease in the level of lamin A/C mRNA.

We hypothesized that naked siRNA was being degraded in the vaginal cavity and/or inefficiently absorbed by the mucosa. To improve the mucosal activity of siRNA, we next focused on using liposomes as carrier vehicles (lipoplexes) given their utility in mediating the cellular delivery of siRNA in vitro¹⁴. To characterize in vivo tissue distribution better, we first used a fluorescent siRNA molecule targeting luciferase. For this series of experiments, we instilled 4 nmol of fluorescein–siRNA lipoplex directly into the vaginal cavity. At 48 h, we euthanized the animals and prepared vaginal tissue for confocal microscopy. As seen in Fig. 1, siRNA was taken up by cells throughout the squamous epithelial layer and also in the submucosa. Within individual cells, siRNA characteristically localized to perinuclear areas, a distribution pattern similar to that seen in cellular transfection experiments in vitro^15,16.

Figure 1.

Confocal microscopy of vaginal tissue in (A) an untreated animal and in (B) an animal treated with a siRNA conjugated to fluorescein. Epithelial and submucosal tissues were examined in four siRNA (4 nmol)-treated animals. Projections of nine optical sections collected at 0.5-m intervals are shown. (Left) Propidium iodide staining, (middle, FITC) fluorescein staining, and (right) merged images. No fluorescein autofluorescence was detected in the control animal. Fluorescent siRNA molecules are seen primarily throughout the squamous epithelial layer and also in the submucosa. Within cells the siRNA is localized to the perinuclear areas (inset). Further digital analysis of fluorescent signal activity revealed that 67% of fluorescent signal emanated from squamous epithelia and the remaining 33% from underlying mucosa.

Full figure and legend (210K)

Given the tissue penetrance of liposomal/siRNA complexes, we next determined the degree and durability of lamin A/C knockdown using this delivery system. We administered 4 nmol of lamin A/C siRNA lipoplex to groups of 10 animals and quantified mRNA levels over a 10-day period. Untreated animals and those treated in the same manner with an irrelevant siRNA or empty lipoplex served as relevant controls. The gene silencing effects by the siRNAs used persisted up to 7 days (Fig. 2A). Maximal gene silencing was achieved and maintained over the first 4 days with lamin A/C mRNA reduced by 86 and 84% on days 2 and 4, respectively. At day 7, we observed a slight diminution of gene silencing with mRNA levels reduced by 79%. By day 10, however, lamin A/C mRNA had returned to pretreatment levels. Neither empty nor irrelevant siRNA lipoplex exerted any effect on the level of lamin A/C mRNA levels compared to untreated or lamin siRNA-treated mice. In a subset of experimental and control animals, we assessed lamin A/C protein and mRNA levels in the cervicovaginal tract by Western and Northern blot, respectively. These results mirrored the RT-PCR results with the selective reduction of protein and mRNA in lamin A/C siRNA-treated animals but not in control animals (Figs. 2D and 2E).

Figure 2.

Target gene expression in siRNA-treated and control animals. Each treatment group consisted of 10 to 15 animals. Target gene mRNA levels were quantified by real-time quantitative PCR, normalized to GAPDH, and then converted to copy number by use of an external standard curve. Copy numbers were then compared to those obtained from untreated animals and are shown in the y axis as percentage expression (SEM) compared to untreated controls. (A) Vaginal application of lamin A/C lipoplex siRNA led to a statistically significant decrease in lamin A/C mRNA expression on days 2, 4, and 7 compared to untreated animals and those control animals treated with liposome only or lipoplex-irrelevant siRNA (^*P < 0.002). No statistically significant differences in lamin A/C mRNA levels were observed among untreated animals or those treated with control preparations. Levels of CCR5 mRNA were quantified in the (B) vagina and (C) rectum after administration of lipoplex CCR5 siRNA, CCR5 antisense, or an irrelevant siRNA. CCR5 mRNA levels were reduced in a statistically significant fashion in CCR5 siRNA-treated animals on day 2 in both rectum and vagina (P < 0.002). Neither antisense, irrelevant siRNA, nor empty lipoplex treatment led to a statistically significant decrease in CCR5 levels. For experiments involving irrelevant siRNA and antisense compounds, levels of targeted genes were quantified 2 days after vaginal/rectal administration. ^*Statistically significant. (D) Western blot of vaginal expression of lamin A/C in four animals treated with 4 nmol of lipoplex lamin A/C siRNA 48 h after administration and two animals treated with an irrelevant siRNA and one untreated animal. siRNA treatment led to the selective reduction of lamin A/C protein in whole vaginal tissue. Following lamin A/C detection, the blots were stripped and probed with antibody to murine GAPDH to control for variations in protein loading. (E) Representative Northern blot of lamin A/C mRNA in vaginal tissue. Lamin A/C siRNA treatment (n = 4) led to a reduction in the levels of corresponding mRNA compared to an untreated animal or those treated with an irrelevant siRNA (n = 2). (F) Western blot of vaginal expression of CCR5 in animals treated with CCR5 siRNA (n = 4) or irrelevant siRNA (n = 2) or an untreated animal (n = 1). CCR5 siRNA treatment led to the selective reduction of CCR5 protein in whole vaginal tissue. Following CCR5 detection, the blots were stripped and probed with antibody to murine GAPDH to control for variations in protein loading.

Full figure and legend (170K)

As our confocal experiments confirmed that siRNA penetrated submucosal vaginal tissue, we reasoned that siRNA molecules were also penetrating mucosal hematopoietic cells such as CD4⁺ lymphocytes and macrophages. To test this rigorously, we designed siRNA targeting the murine CCR5 chemokine receptor. CCR5 expression in mice parallels that of humans with similar levels of expression in lymphocytes and tissue macrophages¹⁷. Once again, we vaginally administered lipoplex CCR5 siRNA to groups of mice and quantified corresponding mRNA over a 10-day period. Untreated mice and those treated with an irrelevant siRNA or a previously characterized CCR5 antisense molecule served as controls. As seen in Fig. 2B, CCR5 mRNA levels were selectively decreased in CCR5 siRNA-treated animals but not in any of the control groups. Similar to our lamin A/C experiments, gene silencing was maximal at day 2 (88%), but thereafter decreased to 61% (day 4), 74% (day 7), and 19% (day 10). We confirmed the specificity of CCR5 siRNA gene silencing by Western blot (Fig. 2F). Given the recent demonstration that lipid formulations widely used to transfect siRNA in vitro can impact gene expression, it should be noted that levels of targeted genes were not decreased in the lipid-only arms that were included in all treatment groups¹⁸.

The efficiency of liposomal siRNA uptake by the predominantly squamous epithelia of the vagina prompted us to determine whether similar levels of uptake and activity were possible in other mucosal surfaces, such as colon, characterized by columnar epithelium. We administered lipoplex CCR5 siRNA to the rectum of animals and compared corresponding mRNA levels among treated and control groups. Compared to control animals, CCR5 mRNA levels were reduced by 72% on day 2 (Fig. 2C). Our ability to target colonic genes prompted us to examine the therapeutic potential of RNAi in the setting of inflammatory bowel disease. Previous work has demonstrated that liposomal solutions of antisense molecules can be effectively delivered to the colonic mucosa. For example, Gao et al. found that a single treatment using an antisense molecule targeting CD40 decreased levels of inflammation in a rat model of colitis¹⁹. Recent studies have demonstrated that pharmacologic reduction of TNF- decreases the severity of disease in animal models and in humans²⁰. We induced acute colitis in mice by continuous feeding with 5% dextran sodium sulfate (DSS). Simultaneously we administered to a subset of animals lipoplex siRNA targeting murine TNF-. We assessed the severity of colitis by blinded review of colonic histology as well as quantification of mucosal levels of TNF- mRNA. Initially we limited our analysis to the descending colon as we hypothesized that lipoplex siRNA preparations were limited in their anatomic localization to the perirectal region after administration by enema. As seen in Fig. 3A, compared to control mice, DSS treatment led to a 58-fold increase in TNF- mRNA levels. In mice receiving TNF- siRNA and DSS, TNF levels were increased by only 5.5-fold. Administration of an irrelevant siRNA had no effect on TNF- mRNA levels (data not shown). Although severe colonic inflammation was evident in all mice treated with DSS, those treated with DSS and siRNA had either mild (n = 6) or moderate (n = 4) inflammatory changes (Fig. 3B). Next, we quantified TNF- mRNA levels in the transverse and ascending colon among siRNA-treated vs untreated control animals receiving DSS. siRNA administration had no significant effect on levels of TNF- mRNA and tissue sections were markedly inflamed in both locations (data not shown). This explained the fact that both experimental and control animals lost 25% of body weight prior to sacrifice and showed frank signs of colitis such as blood-streaked stool. However, the selective reduction of proinflammatory cytokines in the left colon is of translational importance since isolated left-sided colitis in association with inflammation of the rectum (proctosigmoiditis) is a common clinical form of inflammatory bowel disease (especially ulcerative colitis) that is particularly amenable to localized delivery systems in the clinic.

Figure 3.

(A) Levels of TNF- mRNA in the descending colon of mice receiving either continuous DSS (n = 15) or continuous DSS and an application of lipoplex siRNA targeting TNF- at days 0 and 2 (n = 11). All animals received 5% DSS in their sole source of water and were subsequently switched to normal water on day 6 prior to sacrifice at day 8. TNF- mRNA levels were quantified by RT-PCR and are expressed as fold increase compared to untreated mice. TNF- levels were increased by 58-fold in DSS-treated mice but increased by only 5.5-fold in mice treated with siRNA and DSS (^*P < 0.01). (B) Severe colitis (left) in a mouse treated with DSS demonstrating extensive ulceration with complete absence of normal colonic crypts and replacement by granulation tissue covered by a fibrinopurulent exudate. In contrast, colonic mucosa from a mouse treated with TNF- siRNA and 5% DSS 5 days (right) demonstrates only focal mild inflammatory cell infiltration and crypt abscess. H&E stain, original magnification 100 (top) or 200 (bottom). Tissue sections were from the descending colon of animals.

Full figure and legend (307K)

A concern of mucosal drug delivery is the untoward chance that either the drug itself or its mode of delivery may lead to mucosal inflammation. This concern is especially relevant in studies of the cervicovaginal and rectal tract where unexpected inflammatory responses may defeat a therapeutic intervention and render the mucosa more permissive to sexually transmitted infection. To evaluate the safety of our lipoplex preparations we microscopically evaluated vaginal tissue in 10 untreated and 10 lamin A/C siRNA-treated mice. One mouse in the untreated group and two mice in the treated group had signs of vaginal inflammation characterized by a superficial mucosal, primarily neutrophilic, inflammatory infiltrate with scattered epithelial apoptotic bodies. These changes may be observed in the latter stages of the murine estrous cycle. Significantly, no mucosal erosions were seen in any of the siRNA-treated animals. To evaluate more formally the proinflammatory profile of treated and untreated animals, we quantified the mucosal levels of various cytokines (e.g., IL-1, IL-10, TNF-, IFN-) and found no differences among the various treatment and control groups (Supplementary Fig. 1).

While our experiments involving administration of irrelevant siRNAs and liposome-only solutions pointed to the sequence specificity of gene silencing, we were also concerned that our siRNA preparations were eliciting a nonspecific interferon response as has been observed under diverse experimental conditions in vitro. Recent studies have demonstrated that transient transfection of siRNA or vector-mediated transfer of shRNA expression cassettes leads to increased expression of 2',5'-oligoadenylate synthetase (OAS), an interferon target gene that is upregulated by long dsRNA as well as dsRNA-activated proteins such as PKR and its substrate, eIF2. A biochemical dissection of this process has led to the identification of potent immunostimulatory sequence motifs in certain siRNAs^21,22,23,24. Heidel et al. recently reported that the hydrodynamic tail administration of naked siRNA to mice did not lead to a nonspecific interferon response as measured by serum levels of IFN- and IL-12²⁵. We were concerned that mucosal RNAi would elicit an interferon response given that the entire dose of siRNA was being localized to a discrete anatomic locus rather than the entire vascular compartment as with hydrodynamic injection. We first verified that the murine mucosa mounted a stereotypic response to double-stranded RNA by treating the vaginal mucosa directly with polyinosinic acid:polycytidylic acid (poly(I:C)), an analog of dsRNA. As expected, poly(I:C) treatment led to an 130-fold increase in the vaginal expression of murine OAS1, as determine by quantitative RT-PCR compared to untreated controls (Fig. 4A). No increase in OAS1 levels could be detected in animals treated with siRNA. Next, we compared the expression of phosphorylated forms of PKR and eIF2 in the vaginal and rectal mucosa of siRNA and poly(I:C)-treated animals. Poly(I:C) treatment but not siRNA led to increased levels of phosphorylated PKR and eIF2, as determined by Western blot (Fig. 4B). To rule out more systematically interferon upregulation, we assessed levels of >100 interferon-related genes by microarray analysis. siRNA treatment had no appreciable effect upon the levels of interferon-related genes in either the rectal or the vaginal mucosa compared to liposome-only treated animals (data not shown). Thus, using a variety of assays, we found that localized mucosal anti-lamin or anti-CCR5 siRNA administration does not lead to a nonspecific interferon response.

Figure 4.

(A) Relative levels of OAS1 mRNA in vaginal (Vag) and rectal (Rec) mucosa as quantified by real-time PCR in poly(I:C)-treated, siRNA-treated, and untreated control (C) animals. siRNA treatment did not lead to increased expression of OAS1 in vivo. (B) Selective upregulation of phosphorylated forms of PKR and eIF2 in mice treated with poly(I:C) but not in untreated mice or those treated with vaginal or rectal siRNA.

Full figure and legend (177K)

Mucosal drugs must meet several requirements, including a specific mechanism of action, rapid onset of action, and durable effect after a single administration. Our work demonstrates that synthetic siRNA possesses many of these qualities. The duration of mucosal gene silencing is likely governed by the potency of the siRNA molecule itself, the eventual intracellular destruction of siRNA, and the natural turnover of treated cells in the cervicovaginal and rectal tract. As expected, our CCR5 experiments led to less durable silencing compared to lamin experiments. It is likely that decreased levels of CCR5 knockdown after day 2 reflect the trafficking of CCR5-expressing immunoregulatory cells such as NK cells and T lymphocytes out of the mucosa. Nevertheless, regardless of gene target or mucosal location, we found that levels of gene silencing at 48 h (>70%) were superior to those obtained by systemic administration²⁶. Thus, the direct mucosal administration of synthetic siRNA constitutes an additional route of delivery that may have therapeutic potential for a variety of disease states. For example, it is conceivable that microbicidal siRNA could be formulated to decrease vaginal CCR5 expression in humans and thereby emerge as an HIV-1 preventative. The full realization of the therapeutic potential of siRNA will depend upon mucosal delivery systems suitable for humans that maximize siRNA tissue penetrance and the identification and generation of siRNA molecules structurally modified to ensure extended intracellular gene silencing capacity after a single local application^27,28.

Materials and Methods

Animals and nucleic acid preparation/administration

Ten- to twelve-week-old C57BL/6 mice (Harlan, Indianapolis, IN, USA) were used in this study. Standard mouse chow pellets and water were supplied ad libitum. All animal procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital. Murine colitis was induced by continuous feeding with 5% DSS (Research Products International, Prospect, IL, USA).

Poly(I:C) was obtained from Sigma (St. Louis, MO, USA). siRNA targeting murine lamin A/C, CCR5, and TNF- were from Dharmacon (Lafayette, CO, USA). The siRNA sense sequences were lamin A/C, 5'-CUGGACUUCCAGAAGAACAdTdT-3'; CCR5, 5'-CUCUACUUGUCAUGGUCAUdTdT-3'; and TNF-, 5'-GACAACCAACUAGUGGUGCdTdT-3'. The CCR5 antisense sequence was 5'-CCGTTCTGACTTTT-3'²⁹. For experiments involving naked siRNA, siRNA (20, 40, 60 nmol) was resuspended in RNase-free water, OptiMEM (Invitrogen, Carlsbad, CA, USA), or siRNA resuspension buffer (Dharmacon). To create lipoplex siRNA preparations, 2 l Lipofectamine 2000 (Invitrogen) was combined with 48 l OptiMEM and incubated for 5 min, according to the manufacturer's instructions. This solution was then combined with 20 nmol siRNA resuspended in 50 l OptiMEM and incubated at room temperature for 20 min. This solution was immediately administered to vaginas/rectums of anesthetized mice (4 nmol/mouse). All siRNA and antisense preparations were administered once (day 0), with the exception of siRNA targeting TNF-, which was administered on days 0 and 2. Animals were not prepped prior to rectal or vaginal administration. In total, 26 animals were used for the DSS experiments; 15 received DSS only and 11 received DSS and siRNA. Ten to fifteen animals were studied for each time point for the lamin A/C and CCR5 siRNA experiments and relevant controls. Each vaginal/rectal administration consisted of a 20-l solution of liposomal siRNA and was delivered using a P20 pipettor.

Tissue preparation and real-time RT-PCR analysis

The animals were euthanized by CO₂ asphyxiation. An animal feeding tube was immediately inserted into the vagina or rectum for identification and tissues were visualized and harvested. Vaginal/colonic tissue was then divided and snap-frozen in Trizol reagent (Invitrogen) with liquid nitrogen. Tissue samples were batched and homogenized in Trizol using a Polytron machine (Kinematica, Bethlehem, PA, USA) on wet ice. Total RNA was extracted using Trizol, according to the manufacturer's instructions. One microgram of extracted RNA from tissue samples was added to reverse transcription reactions as described elsewhere³⁰. PCR was performed with 1 l cDNA in a final volume of 25 l containing 1 Qiagen PCR buffer (Qiagen, Valencia, CA, USA), 10 pmol of each primer (lamin A/C, Lam-S, 5'-GAGAGGCTAAGAAGCAGC-3', and Lam-AS, 5'-ACGCAGTTCCTCGCTGTAA-3'; 2',5'-OAS1, OAS-S, 5'-TGGAAAGAAGAGGTCCTGGA-3', and OAS-AS, 5'-ACGGTGCCATTCCCAAAGCA-3'), 0.2 mM dNTPs, SYBR Green I (Molecular Probes, Eugene, OR, USA) (1:750,000), 10 nM fluorescein, and 1.25 units Q-HotStartTaq (Qiagen). To normalize the samples for absolute RNA amounts a GAPDH-PCR was performed with murine primers murgap-1, 5'-TTCACCACCATGGAGAAGGC-3', and murgap-2, 5'-GGCATGGACTGTGGTCATGA-3'. Real-time quantitative PCR was carried out in an iCycler (Bio-Rad, Hercules, CA, USA) using the following thermal cycling profile: 95°C for 15 min, followed by 40 cycles of amplification (95°C for 15 s, 63°C for 30 s, 68°C for 30 s.) All samples were run in duplicate. To allow conversion of real-time PCR-generated threshold cycles to relative copy number, an external standard curve was generated by using a serially diluted plasmid DNA standard containing lamin A/C, CCR5, TNF-, or OAS1 cDNA.

Confocal microscopy

Whole, freshly excised murine vagina specimens were placed in RPMI medium (ATCC, Manassas, VA, USA), and approximately 0.5-cm transverse sections were cut. Sections were mounted on aluminum specimen mounts, in TBS Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC, USA), oriented, and then frozen in liquid nitrogen. Frozen sections of 6 m thickness were mounted on Fisherbrand Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA, USA) and immediately fixed in acetone for 2 min. After a brief rinse in 50:50 acetone:water and two changes of water, slides were coverslipped using Vectashield Mounting Medium containing propidium iodide (Vector Laboratories, Burlingame, CA, USA). Confocal images were acquired with a Nikon PCM 2000 (Nikon, Inc., Mellville, NY, USA) using the argon (488) and the green helium–neon (543) lasers. Serial optical sections were performed with Simple 32, C-imaging computer software (Compix, Inc., Cranberry Township, PA, USA). Z series sections were collected at 0.5 m with a 60 PlanApo lens and a scan zoom of 1 or 2. Images were processed and reconstructed in NIH Image shareware (National Institutes of Health, Springfield, VA, USA). Adobe PhotoShop was used to convert the images to RGB and in the assembly of figures. To quantify the anatomic localization of fluorescent siRNA in epithelial and submucosal tissue, 12-bit images were acquired with a Spot II digital camera (Diagnostic Instruments, Sterling Heights, MI, USA) using a Nikon E800 microscope (Nikon) and a 40 PlanApo objective. All acquisition parameters were kept the same for all images and intensity measurements were made with IPLab (Scanalytics, Inc., Fairfax, VA, USA).

Western blot

Protein concentrations were determined from Trizol-treated and homogenized vaginal and rectal tissue using the RC DC protein assay (Bio-Rad). Twenty micrograms of protein per lane was resolved on 12% SDS–polyacrylamide gels and transferred onto Hybond PVDF membranes (Bio-Rad). For sequential probing, blots were stripped in Restore Western Blot Stripping Buffer (Pierce, Rockford, IL, USA). Polyclonal antibodies against murine lamin A/C (BioVision, Mountain View, CA, USA) and murine CCR5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at a 1:1000 dilution. A rabbit monoclonal antibody to murine GAPDH (Abcam, Cambridge, MA, USA) was used at a 1:2000 dilution. The monoclonal PKR antibody (Santa Cruz Biotechnology) was used at a 1:500 dilution; anti-eIF2 (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-eIF2 (Ser51) (Cell Signaling Technology), and anti-phospho-PKR (T451) (Abcam) antibodies were used at a 1:1000 dilution. Immunodetection was performed using the peroxidase-based ECL Plus detection system (Amersham) according to the manufacturer's instructions.

Northern blot

Ten micrograms of total RNA was loaded on a 1% denatured agarose gel and Northern blot was performed with a NorthernMax kit (Ambion). A Zeta-Probe GT membrane (Bio-Rad) was used for transfer. Murine lamin sense oligonucleotide (5'-GAGAGGCTAAGAAGCAGCTTCAGGACCTGTCTC-3') was [-³²P]UTP labeled using the mirVana miRNA probe construction kit (Ambion). Hybridization was carried out at 42°C. Membranes were washed twice in 2 SSC, 0.1% SDS and 0.2 SSC, 0.1% SDS at 37°C and exposed to X-ray film at -80°C.

Statistical analysis

Differences between treatment groups were evaluated by one-way ANOVA with significance testing by Bonferroni-adjusted t tests using the data analysis tools of Microsoft Excel.

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Appendices

Appendix A

Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymthe.2006.04.001.

Acknowledgements

This work was supported by a Career Development Grant and R01AI058697 from NIAID/NIH (B.R.), the Center for Cancer Research Development (NIHP20RR017695), and the Roger Williams Medical Center COBRE (NIHP20RR018757). This research has been facilitated by the infrastructure and resources provided by the Lifespan/Tufts/Brown Center for AIDS Research (NIHP30 AI42853).