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Researchers Develop Molecular (gene therapy) Microbicide Protection Against HIV Infection: put gene therapy into CD4s in mice & this prevented HIV transmission
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study team applied therapy CD4-AsiCs topically within the vaginal canal of female mice with humanized immune systems, and then exposed those mice intravaginally to HIV so as to mimic sexual transmission of the virus. As in the in vitro model, the CD4-AsiCs were able to penetrate through the vaginal walls of these mice to the immune cells within the tissues, deliver the siRNAs to cells displaying CD4, and turn off the expression of the targeted genes. Over the following 12 weeks, none of the mice treated with the siRNAs showed any biological signs of HIV infection, while all of the control mice progressed to full-blown HIV infection...... Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.
Lieberman thinks that the RNAi-based microbicide's specificity and duration of action make it attractive for further pharmaceutical development....According to Wheeler, the method's modularity suggests that its promise is not limited to HIV. "You could basically switch in or out any kind of siRNA or aptamer for any binding target to knock down any gene you would want, be it host or viral." Lieberman added, "Conceivably, one could include siRNAs against multiple viral agents in a cocktail to gain protection from multiple sexually transmitted diseases, including HSV and human papilloma virus."
Using a technique that silences genes promoting infection, researchers have developed a novel, topically-applied molecular microbicide capable of preventing HIV transmission. The microbicide is predicted to have long-lasting effects in mice, opening the door to developing an intravaginal microbicide that could protect women against HIV infection potentially for weeks at a time and bolster public health efforts to halt the spread of HIV/AIDS.
The microbicide takes advantage of a molecular phenomenon called RNA interference (RNAi), in which small pieces of RNA called small interfering RNAs (siRNAs) silence the expression of individual genes with complementary sequences. Originally observed in plants, RNAi was found to be active in mammals only a decade ago, but it is already the focus of many clinical investigations.
The researchers, led by Lee Adam Wheeler and Judy Lieberman, chose to investigate RNAi's potential to provide a molecular barrier against HIV transmission based on earlier work in her laboratory showing that the phenomenon could be harnessed to prevent herpes simplex virus (HSV) transmission, and also on recent advances in understanding how HIV penetrates the body. "The current model of HIV transmission holds that the virus is localized to the genital tract for about a week, which could provide a window of opportunity to intervene and prevent the infection from establishing itself throughout the body," said Lieberman. "And last year it was shown that it is possible to prevent HIV transmission, at least to some extent, with a topical vaginal agent using an antiviral drug, thus providing proof-of-principle that a topical strategy could interfere with virus transmission."
In the current study, the researchers used siRNAs that turned off two viral genes and that of one of HIV's two host co-receptors, CCR5. HIV uses CCR5, found on immune cells called T cells and macrophages, to gain entry into an uninfected person's immune cells and establish a foothold within the body. Individuals harboring mutations that deactivate CCR5 are resistant to infection with HIV.
To ensure that the siRNAs would be delivered only to the immune cells targeted by HIV, the research team linked the siRNAs to an aptamer - a second piece of RNA designed to attach to a specific molecule - that binds to HIV's main receptor, CD4, to create CD4 aptamer-siRNA chimeras (CD4-AsiCs).
"By using CD4 as a binding site but knocking down CCR5, we get specificity for the cells targeted by HIV but avoid the risk of interfering with the overall immune response," Lieberman noted.
When tested in vitro using cell lines and blood cells, the CD4-AsiCs bound only to immune cells displaying CD4 on their surface; turned off expression in those cells of the three targeted genes; and prevented HIV replication. In addition, CD4-AsiCs successfully penetrated cultured human cervicovaginal tissues to reach immune cells deep within the tissue layers, silence target gene expression, and prevent HIV infection of the cultures.
To test the effectiveness of this system in vivo, the study team applied CD4-AsiCs topically within the vaginal canal of female mice with humanized immune systems, and then exposed those mice intravaginally to HIV so as to mimic sexual transmission of the virus. As in the in vitro model, the CD4-AsiCs were able to penetrate through the vaginal walls of these mice to the immune cells within the tissues, deliver the siRNAs to cells displaying CD4, and turn off the expression of the targeted genes. Over the following 12 weeks, none of the mice treated with the siRNAs showed any biological signs of HIV infection, while all of the control mice progressed to full-blown HIV infection.
Lieberman thinks that the RNAi-based microbicide's specificity and duration of action make it attractive for further pharmaceutical development. "The problem with most topical methods for preventing sexual transmission of disease is that you have to use them just before having sex, and compliance is a huge issue," she said. "But our laboratory results show that we can knock down CCR5 expression potentially for weeks, suggesting that we could create a stable viral-resistant state where one would only have to apply the agent every couple of weeks."
According to Wheeler, the method's modularity suggests that its promise is not limited to HIV. "You could basically switch in or out any kind of siRNA or aptamer for any binding target to knock down any gene you would want, be it host or viral." Lieberman added, "Conceivably, one could include siRNAs against multiple viral agents in a cocktail to gain protection from multiple sexually transmitted diseases, including HSV and human papilloma virus."
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Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras - pdf attached
Download the PDF here
J Clin Invest.May 16 2011
Lee Adam Wheeler1,2, Radiana Trifonova1, Vladimir Vrbanac3, Emre Basar1, Shannon McKernan1, Zhan Xu1, Edward Seung3, Maud Deruaz3, Tim Dudek4, Jon Ivar Einarsson5, Linda Yang6, Todd M. Allen4, Andrew D. Luster3, Andrew M. Tager3, Derek M. Dykxhoorn1,7 and Judy Lieberman1
1Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA.
2MD-PhD Program (Immunology), Harvard Medical School, Boston, Massachusetts, USA.
3Massachusetts General Hospital, Boston, Massachusetts, USA.
4Ragon Institute of MGH, MIT, and Harvard, Boston, Massachusetts, USA.
5Brigham and Women's Hospital, Boston, Massachusetts, USA.
6Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
7University of Miami Miller School of Medicine, Miami, Florida, USA.
Address correspondence to: Judy Lieberman, 200 Longwood Avenue WAB 255, Boston, Massachusetts 02115, USA. Phone: 617.713.8600; Fax: 617.713.8620; E-mail: lieberman@idi.harvard.edu.
ABSTRACT
The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.
Introduction
The CAPRISA004 study, which demonstrated partial protection from sexual transmission of HIV-1 by vaginally applied tenofovir gel (1), has galvanized interest in developing an HIV microbicide. One of the major obstacles confronting this and other strategies for interrupting transmission is the transience of protection, requiring topical application just before sexual exposure and raising associated problems with compliance (2). Soon after RNAi was found to operate in mammalian cells, multiple groups showed that RNAi could be harnessed to inhibit HIV infection in vitro (3-7). Moreover, siRNAs directed against conserved viral gene sequences or the HIV receptor or coreceptor inhibit diverse viruses from multiple clades (3, 8, 9). Although knocking down the HIV receptor CD4 inhibits HIV transmission (3), targeting the CD4 gene would likely interfere with mounting effective immune responses and is therefore not desirable. CCR5, the HIV coreceptor responsible for virtually all sexual transmission of HIV (10, 11), is a more attractive RNAi target. CCR5 antagonists (12) have already proven useful at preventing HIV transmission in nonhuman primates (13) and humans (14-17). Humans bearing homozygous CCR5 mutations that abrogate CCR5 function are resistant to HIV infection and do not lead to any significant immune dysfunction (18-22). siRNAs directed against CCR5 efficiently silence gene expression for several weeks in vitro in nondividing macrophages, which suggests that gene knockdown might be used to induce durable resistance to HIV infection, circumventing the need to apply a microbicide just before each sexual encounter (8). In fact, sexual transmission of another virus, herpes simplex virus type 2 (HSV-2), can be blocked in mice for at least a week by intravaginal (IVAG) application of siRNAs targeting HSV-2 genes and the HSV-2 receptor, nectin-1 (23, 24).
Translation of these promising results for blocking HSV-2 transmission to HIV prevention, however, must first overcome the hurdle of in vivo siRNA delivery to the immune cells that HIV infects, principally CD4+ T cells and macrophages, which are resistant to most transfection techniques. Although cholesterol-conjugated siRNAs are efficiently taken up by epithelial cells throughout the genital tract (including deep in the lamina propria, resulting in protection against lethal HSV-2 infection in mice; ref. 24), these reagents do not knock down gene expression in T lymphocytes or macrophages when applied IVAG to mice (E. Basar, unpublished observations). We previously developed a method for cell-specific siRNA transfection of immune cells that uses a fusion protein composed of a cell-targeting antibody fragment joined to a protamine peptide that binds nucleic acids (25, 26). siRNAs mixed with the fusion protein are taken up by and knock down gene expression in cells bearing the cognate surface receptor, both in vitro and in tissues after intravenous injection. Modifications of this approach effectively inhibit HIV infection in humanized mice (27). However, antibody-based fusion proteins are expensive to manufacture, are potentially immunogenic, and may require refrigerated storage, making then ill-suited for use in a microbicide for resource-poor settings.
Chimeric RNAs, composed of an siRNA fused to an aptamer (a structured RNA selected to bind a cell surface ligand with high affinity), provide an attractive alternative for in vivo gene knockdown (28-31). Aptamer-siRNA chimeras (AsiCs) efficiently transfect and knock down gene expression in cells bearing the surface receptor recognized by the aptamer. Intravenous injection of AsiCs incorporating aptamers targeting prostate surface membrane Ag (PSMA) silence target gene expression in orthotopic prostate cancer mouse xenografts (28, 29). AsiCs containing an aptamer that recognizes HIV-gp120 inhibit HIV replication in already infected cells in vitro (30, 31) and in vivo (32). However, to prevent HIV transmission, it might be better to inhibit de novo infection of uninfected cells. Since HIV only infects cells bearing the CD4 receptor, CD4 AsiCs (CD4-AsiCs) could, in principle, inhibit infection of all the cells that HIV infects. To test the ability of CD4-AsiCs to inhibit HIV transmission, we engineered AsiCs using 2 high-affinity CD4 aptamers that selectively bind to human, but not mouse, CD4 (33). CD4-AsiCs might inhibit HIV infection in 2 ways: by blocking viral entry via binding to CD4 and by RNAi knockdown of viral genes, host receptors, or other host genes required for viral replication. Here we showed that CD4-AsiCs bearing siRNAs targeting HIV gag and vif or host CCR5 were specifically taken up by CD4+ cells; knocked down expression of their intended target genes; and inhibited HIV infection in primary CD4+ T cells and macrophages in vitro, in polarized cervicovaginal explants, and in immunodeficient NOD/SCID Il2rg-/- (NSG) mice reconstituted with human fetal liver CD34+ cells and surgically implanted with human fetal thymic tissue (so-called "BLT mice"; ref. 34). Although the CD4 aptamer on its own inhibited HIV infection to some extent, chimeric RNAs were significantly more effective at preventing transmission to cervicovaginal explants and to BLT mice.
Discussion
Delivery remains a significant obstacle to the clinical development of siRNA-based drugs. Although cholesterol-conjugated siRNAs silence gene expression without apparent toxicity in mucosal epithelial cells and can be used to prevent HSV-2 transmission (24), this approach would not inhibit HIV transmission, since cholesterol-conjugated siRNAs do not transfect the cells that HIV infects. siRNAs can be delivered into immune cells by receptor-mediated endocytosis, by either complexing siRNAs to antibody fusion proteins or encapsulating siRNAs into liposomes or other nanoparticles bearing targeting antibodies or ligands to cell surface receptors (25-27, 45). Here we demonstrated that an alternate approach - chimeric RNAs composed of an aptamer linked to an siRNA - delivered siRNAs and knockdown target gene expression specifically in primary CD4+ T cells and macrophages, regardless of activation state, in vitro, in intact human explants and in humanized mice. Importantly, the CD4-AsiCs were able to inhibit vaginal HIV transmission to humanized mice. Although only 2 of 4 CD4-AsiC-treated mice maintained undetectable virus throughout the 12-week period, the transient viremia, detected only after 7-9 weeks after infection, was just above the limit of detection. All CD4-AsiC-treated mice showed preserved T cell counts, and none had measurable antigenia at any time. These promising results were achieved without any optimization of the CD4-AsiCs for CD4 binding or gene silencing sequences, using an extremely high challenge virus dose that gave uniform infection of control mice. It may be easier to prevent sexual HIV transmission in humans, which is very inefficient, requiring hundreds of exposures for each transmission event, and where usually only a single virion is able to establish a foothold in the host (46). Protection was achieved here with a highest dose of approximately 0.2 mg/kg (120 pmol), which is a feasible dose for small RNA drugs, but which might be reduced even further by drug optimization. Future studies in humanized mice will evaluate how long gene silencing in the genital tissue and protection lasts in order to determine whether RNAi-based microbicides could be intermittently dosed with acceptable compliance.
Targeted delivery has the potential dual advantage of reduced toxicity to bystander cells and reduced effective dose. What delivery approach is preferable for clinical or research purposes is difficult to determine ab initio and may depend on the application. Chimeric RNAs have the advantage of being a single molecule rather than a complex mixture, are less likely to be immunogenic than proteins, and are more straightforward to purify and less costly to produce than RNAs that need to be formulated with proteins, nanoparticles, or liposomes. CD4-AsiCs were shown to knock down 2 viral genes, 1 transgene (luciferase), and 4 host genes (CCR5, CD45, lamin A, and EG5) and can likely be designed to inhibit the expression of any gene. The kinetics of target gene suppression may differ between targets, depending on target gene mRNA and protein stability. For example, CCR5 and CD45 surface protein expression was not appreciably reduced until 72 hours after CD4-AsiC treatment. However, mRNA levels of these genes, when measured by qRT-PCR, declined within a day.
This study builds on previous studies that used intravenous injection of PSMA-AsiCs to transfect human prostate cancer cells in an orthotopic mouse tumor model (28, 29) or that used HIV gp120-AsiCs to transfect HIV-infected CD4+ cells in vitro and in vivo (30-32). Based on these studies, the CD4-AsiCs developed here likely could be used for systemic gene silencing in circulating immune cells. If CD4-AsiCs perform as well as we expect, they may be a potent tool for genetic manipulation (which has been so powerful for understanding mouse immunology by use of knockout mice) to study the role of individual molecules in complex human immune responses in humanized mice in vivo. Because CD4-AsiCs do not appear to perturb CD4 cell surface expression or alter other immune receptors that are sensitive indicators of immune activation, it should be possible to test the effect of knocking down one gene product at a time. The lack of cellular toxicity of CD4-AsiCs might also make them an attractive alternative to electroporation for in vitro transfection of CD4+ T cells. The clone 9 aptamer also recognizes rhesus macaque CD4, and the corresponding CD4-AsiC knocks down gene expression in rhesus PBMCs in vitro (L.A. Wheeler, unpublished observations), which suggests that CD4-AsiCs could also be used to study SIV or SHIV infection and immune responses in nonhuman primates.
The micromolar concentrations of CD4-AsiCs used here for in vitro gene knockdown and HIV infection inhibition studies are somewhat higher (about 2-10 times more) than those published in some previous reports (3, 24, 28, 31). The higher concentration could be due to differences in target cell type. The CD4-AsiCs have not been optimized, and improvements in CD4-AsiC design or synthesis could increase silencing efficiency. Modifications might include optimizing the aptamer or siRNA sequence, altering the linker joining the aptamer to the siRNA, interchanging the 2 siRNA strands, or replacing the double-stranded siRNA with a stem-loop that mimics endogenous miRNA structures. Some of these changes have been used in earlier studies, where the optimal AsiC construct depended on the particular siRNA sequence (29, 31). Studies are in progress to understand how CD4-AsiCs are taken up and delivered to the cytosol, where they interact with the endogenous RNAi machinery. To optimize CD4-AsiC design, it will be helpful to compare not only the efficiency of gene silencing, but also the efficiency of intracellular processing to the mature siRNA and its incorporation into the RNA-induced silencing complex. It may also be helpful to shorten the construct to make chemical synthesis more practical. We plan to test whether any part of the aptamer sequence can be deleted without losing binding affinity. Another approach would be to synthesize the aptamer and siRNA separately, using complementary adapter sequences to join them (30, 31).
Despite the high concentrations required in vitro, CD4-AsiC-mediated in vivo silencing and protection from HIV infection required significantly lower doses (about 7-25 times less) than were used to inhibit HSV-2 transmission with lipoplexed or cholesterol-conjugated siRNAs (23, 24). The stability of the CD4-AsiCs over 36 hours in human vaginal fluid suggests that further stabilization for topical use may not be required. However, the in vivo half-life in the blood and other body fluids and within cells, as well as the efficiency and durability of gene knockdown, might be improved by further chemical modifications, such as introduction of 2'-OCH3 to purines on the active strand. For systemic use, chemical conjugation to cholesterol or polyethylene glycol might also improve circulating half-life (29, 36).
The pathways used to deliver RNAs into cells, whether by aptamers or by other delivery methods, remain poorly understood. Our preliminary studies suggest that Cy3-labeled CD4-AsiCs are initially taken up into early endosomes and then escape to the cytosol. Endocytosis might be triggered by activation of the CD4 receptor by aptamer binding or occur via the continuous basal internalization of cell surface receptors. The latter pathway may be more likely, since CD4 cell surface expression is not appreciably altered by CD4-AsiC treatment and since CD4-AsiCs are monomeric and are not expected to crosslink the receptor. Although lack of perturbation of CD4 surface expression would be ideal for using CD4-AsiCs as a research tool, a divalent or polyvalent reagent that activates temporary CD4 internalization might also have advantages for HIV prevention or therapy. This might add a third mechanism for inhibiting HIV cellular transmission (removal of the viral receptor from the cell surface) to the other 2 mechanisms demonstrated in the present study (gene silencing and partially blocking the virion binding site on CD4). Once in the endosome, how the RNA is released into the cytosol is unknown. Understanding the uptake and trafficking within cells of CD4-AsiCs will undoubtedly facilitate design improvements.
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