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Recent Meeting with NCI Director Dr. John Niederhuber Highlights the History, Strategy, and Successes of HIV/AIDS Research at the CCR
n November 6, 2006, NCI Director John Niederhuber, MD, met with several of CCR’s leading scientists conducting research in HIV/AIDS and AIDS-associated malignancies to discuss the advances they have been making in the laboratory and the clinic. NCI’s Intramural Research Program (IRP) has played a leading role in HIV/AIDS research for almost 25 years. In this article, which is based on discussions from the meeting with Dr. Niederhuber, I briefly review the history of HIV/AIDS research in the IRP, describe the important role that IRP scientists play in this area of research today, elucidate CCR’s strategy for HIV/AIDS research, and summarize current research efforts and the tremendous gains our scientists are making in these investigations. History Robert Yarchoan, MD, Chief of the HIV and AIDS Malignancy Branch, opened the meeting by providing background information on HIV/AIDS research in the IRP. As Dr. Yarchoan noted, one of the first signs of the AIDS epidemic was an outbreak of Kaposi’s sarcoma in young men; indeed, AIDS would go on to fuel an epidemic of certain cancers. In response to this, several NCI researchers immediately turned their attention to this new disease. At the onset of the AIDS epidemic, IRP investigators possessed critical expertise in several key areas that allowed them to address this public health crisis:
In part, as a result of this expertise, IRP scientists were able to make quick, substantive advances in the emerging HIV/AIDS crisis in the early 1980s. Without their contributions, progress in HIV/AIDS research would not be as advanced as it is today. In 1984, just three years after the first AIDS patient was admitted to the Clinical Center (Metabolism Branch), the group led by Robert Gallo, MD, then at the NCI’s Laboratory of Tumor Cell Biology, and a team at the Pasteur Institute in France were able to identify the retrovirus HIV as the causative agent of AIDS. That same year, the first diagnostic blood test for HIV infection was developed at the NCI. Soon thereafter, Samuel Broder, MD, Robert Yarchoan, MD, and Hiroaki Mitsuya, MD, PhD, began to seek effective therapy for AIDS, and discovered or co-developed the first effective drugs—AZT (zidovudine), ddC (zalcitabine), and ddI (didanosine). In particular, this group identified the anti-HIV activity of these drugs and, with support from other NCI components, conducted the initial clinical trials. These drugs would become the first U.S. Food and Drug Administration (FDA)–approved anti-HIV drugs. Today, they are combined with other agents to form a treatment called highly active antiretroviral therapy (HAART). Since they were first introduced, AIDS therapies are estimated to have saved at least 3 million life-years in the United States (Walensky RP et al. J Infect Dis 194: 11–19, 2006) and have reduced the burden of HIV-associated cancers such as Kaposi’s sarcoma and certain lymphomas. This kind of research productivity continues to this day. The CCR currently has one of the largest and most productive HIV/AIDS research programs in the world.
During the meeting, Dr. Niederhuber noted that cancer research is a model for research in many other areas and that there are common threads between cancer and AIDS. During his presentation, Dr. Yarchoan expanded on this theme by explaining the interrelationships between research on AIDS-associated malignancies and research on HIV itself. He explained that there is a unity to research on all retroviruses, including those that cause cancer and AIDS. At the translational level, anti-HIV therapy is much like cancer therapy. Both involve multidrug therapies, long-term treatment, and problems of toxicity and drug resistance; these particular similarities stem from the fact that HIV infection and cancer are both chronic diseases that evade and/or suppress the immune system. Jay Berzofsky, MD, PhD, of the Vaccine Branch, noted that “Because of these parallels, there is much opportunity for cross-fertilization between the two fields of study.” For more than two decades, the intramural program has made many contributions to HIV/AIDS research, providing new ideas, fresh approaches, and a long history of successes, ranging from the establishment of diagnostic tools to drug development and clinical treatments. HIV/AIDS research evolved naturally in the IRP because of the retroviral work already being performed in the program prior to the onset of the AIDS epidemic. Today, the CCR has one of the greatest concentrations of scientists combating HIV/AIDS. Our investigators have broad, complementary, and synergistic expertise that they direct toward a better understanding of this disease, and continually provide abundant returns on the investments made in CCR's HIV/AIDS research programs. Interactions with the Extramural Community IRP investigators are encouraged to pursue high-risk research that can have a major impact, but which may be too difficult or risky for academia or industry. However, they have forged fruitful partnerships with extramural scientists as well as investigators in other institutes of the NIH. A large part of the success of these partnerships stems from the resources our investigators have shared with the other researchers. Stuart Le Grice, PhD, of the HIV Drug Resistance Program (DRP) Resistance Mechanisms Laboratory, noted that the large-scale virus culture facilities of the NCI-Frederick AIDS Vaccine Program, which were critical to the initial development of diagnostic kits to screen the blood supply for HIV-1 two decades ago, have since provided an invaluable source of highly purified virus for both basic and applied research. Intramural investigators have long been prolific and generous providers of reagents for HIV research. These materials have been shipped worldwide to academic institutions, industry, and governmental agencies. Although some reagents have been licensed to commercial enterprises, most are provided at no charge to qualified scientists trying to develop more effective ways to treat HIV/AIDS. Additionally, intramural scientists have developed and shared highly sensitive methods of HIV detection for analyses of patient samples from a variety of clinical studies. They have also given others free access to the databases they have developed and maintained over the years, which continue to serve as important sources of information for many scientists, educators, and students. Strategy, Investigations, and Recent Successes The CCR has developed an infrastructure that allows flexibility and fosters innovation. Although basic research is a substantial part of the CCR’s work, the Center has as strong a commitment to translational and clinical research. Basic, translational, and clinical scientists form multidisciplinary teams that are pursuing new approaches for the prevention and treatment of HIV/AIDS. CCR investigators have been integrating discoveries made in basic research laboratories and in animal and tissue models with clinical research and are working to produce new drugs and technologies that will improve the lives of patients with HIV/AIDS. The principal areas of focus of HIV/AIDS research at the CCR are drug resistance and drug development, vaccines and immunotherapy, AIDS-associated malignancies, and translational and clinical research in these areas. Specific examples of the high-impact research being performed by CCR investigators were presented to Dr. Niederhuber and are briefly described below. Drug Resistance and Drug Development Stephen Hughes, PhD, of the HIV DRP Retroviral Replication Laboratory, discussed efforts to develop new drugs and drug strategies that will be effective against both wild-type HIV-1 and the known drug-resistant mutants. He described ongoing efforts within the CCR to better understand the structures of two key drug targets—integrase (IN) and reverse transcriptase (RT)—and to better understand the mechanisms by which RT develops resistance to the available drugs. Data from these studies have been made available to intramural and extramural researchers and are being used to develop new and more effective inhibitors of HIV-1. Dr. Hughes also described complementary efforts within the CCR to screen and develop drugs that work against new targets, such as RT-associated RNaseH activity and the viral structural protein Gag. Panacos Pharmaceuticals, working with CCR scientists, has developed a novel inhibitor that blocks a specific step in the maturation of Gag. The inhibitor is currently in late-stage clinical trials. Vineet KewalRamani, PhD, also of the HIV DRP Retroviral Replication Laboratory, discussed his work in developing a new macaque model that can be used to test anti-HIV therapies and to investigate the cellular origins of HIV reservoirs in humans. The new model uses RT-SHIV, a chimeric virus that is based on a simian immunodeficiency virus (SIV) and encodes a key drug target, RT, from HIV-1. Unlike SIV, RT-SHIV is susceptible to a commonly employed HAART. Dr. KewalRamani is using the model to screen for the virus-infected cells that persist during HAART. He also noted that the model has the potential to help answer critical questions about the development of antiviral drug resistance and will be a valuable tool for the development and evaluation of new drugs.
Dr. Berzofsky gave an overview of the challenges in vaccine development, such as HIV strain diversity and rapid mutation rate, latent reservoirs of virus, lack of CD4 T-cell help, and the difficulty with HIV envelope structure for inducing broadly neutralizing antibodies. He also summarized results indicating the need for high-avidity T cells to clear virus infection and for mucosal immunity, both to prevent mucosal transmission and to clear the major reservoir of virus in the gut mucosa, based on data from his lab and the Vaccine Branch. He noted that CCR scientists are well positioned to address these challenges because of expertise in cancer vaccines, T-cell immunology, mucosal immunology, and retrovirology. Marjorie Robert-Guroff, PhD, also of the Vaccine Branch, described her work in developing a replication-competent adenovirus-HIV recombinant vaccine that has mucosal tropism for induction of mucosal immunity, critically important for preventing HIV infection. This live, replicating vaccine elicits enhanced immune responses compared to current non-replicating vaccine candidates and has demonstrated potent protective efficacy in preclinical studies. Dr. Robert-Guroff is establishing a phase I trial of oral, replication-competent adenovirus-4-HIV env in HIV-negative volunteers, to be conducted at the Clinical Center in collaboration with the National Institute of Allergy and Infectious Diseases (NIAID). Jeff Lifson, MD, of the AIDS Vaccine Program, summarized research on a novel vaccine approach based on chemically inactivated virions that retain structurally and functionally intact envelope glycoproteins on their surfaces. These virions, developed as an outgrowth of basic research findings by AIDS Vaccine Program investigators, are being extensively used as reagents by hundreds of investigators for a variety of research applications. Dr. Lifson and his colleagues are studying their potential as vaccine immunogens in non-human primate models and are working with leading clinical investigators in the United States and Europe to establish an early phase clinical trial. Clinical Applications/AIDS-associated Malignancies John Coffin, PhD, of the HIV DRP, discussed recent advances in the area of clinical research. He discussed the development, in the HIV and AIDS Malignancy Branch, of combinations of drugs containing interleukin-12 as novel therapies for Kaposi’s sarcoma. He then noted that DRP investigators have devised highly sensitive assays that can be used to measure HIV levels in plasma less than 1 virion per mL and to determine the frequency of drug-resistant mutants in infected patients. These investigators have been working closely with collaborators in the NIAID HIV clinical program and at academic institutions and pharmaceutical companies worldwide to apply these assays to patient samples from a variety of clinical studies. DRP investigators have also shown that treating South African women with a single dose of the non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine (to prevent mother-to-child transmission) induced persistent NNRTI-resistance mutations in approximately 70% of the study subjects. Dr. Coffin stated that the potential of these mutations to compromise further treatment options for women treated with this agent is a serious but, as yet, poorly understood issue. Mary Carrington, PhD, of the Laboratory of Genomic Diversity, summarized her
research with the chemokine receptor gene CCR5, which encodes a protein that serves
as the major cellular co-receptor for HIV-1. CCR investigators were among the first to identify a 32–base pair deletion in the coding region of this gene that protects against HIV-1 infection in people who are homozygous for the mutation and slows AIDS progression in heterozygous individuals. This was the first major success in the identification of a genetic variant that affects the outcome of HIV-1 exposures and infections. Dr. Carrington noted that in some cases, genetic studies have guided functional work, and that the design of drug therapies and vaccines against HIV has been based partly on genetic studies. Dr. Niederhuber’s goal for the meeting was principally to have a dialogue with key researchers about the basic, translational, and clinical research over the last two decades to combat HIV/AIDS. He also wanted to express his appreciation for CCR’s HIV/AIDS and AIDS malignancy research programs and to stress their importance to the Institute. The CCR has a rich and proud history of HIV/AIDS and AIDS-associated malignancies research. We have continuously proven ourselves to be a powerful force in the worldwide effort to resolve this growing health crisis, and we look forward to future successes. We are very grateful to Dr. Niederhuber for taking time from his busy schedule to have a scientific discussion with us, and we are particularly grateful for his continued support of our HIV/AIDS and AIDS malignancy research programs.
TRAF2 Plays a Key, Non-redundant Role in LIGHT/Lymphotoxin β Receptor Signaling
umor necrosis factor (TNF)–related cytokines play critical roles in inflammatory and immune responses. Acting through their specific cellular receptors, these cytokines initiate diverse signaling pathways that regulate cell death, survival, and differentiation. LIGHT is one member of the TNF superfamily, and its receptor, lymphotoxin β receptor (LTβR), belongs to the TNF-receptor superfamily. LTβR is a key mediator for development, organization, and differentiation of lymphoid tissue and is expressed on most types of cells including fibroblasts and epithelial and myeloid lineages. The pathway involving LIGHT/LTβR–mediated activation of transcription factor nuclear factor-κB (NF-κB) is a major pathway responsible for LIGHT and LTβR’s diverse biological functions. Previous studies have suggested that TNF receptor–associated factors, such as TRAF3 and TRAF5, are key mediators of LTβR signaling, particularly in the activation of the NF-κB pathway. We found that LIGHT activates both NF-κB and c-JUN N-terminal kinase (JNK), to control the life and death of a cell. NF-κB regulates the expression of genes crucial to innate and adaptive immune responses, cell growth, and apoptosis. JNK, also known as stress-activated kinase (SAPK), is activated by many apoptosis-inducing stimuli and is thought to be an important apoptotic mediator. In addition, JNK activation is involved in many other biological processes, such as cell proliferation, embryogenesis, and immune responses. We demonstrated that TNF receptor–associated factor-2 (TRAF2) has an essential, non-redundant role in LIGHT/LTβR–mediated activation of both NF-κB and JNK. In HeLa cells, LIGHT induces NF-κB and JNK activation, which can be blocked by the dominant-negative mutant of TRAF2. We found that LIGHT treatment results in the recruitment of TRAF2, TRAF3, and IKK (a protein kinase) to the LTβR signaling complex. Although both NF-κB and JNK pathways are activated by LIGHT in wild-type mouse embryonic fibroblasts (MEFs), neither of these two pathways was activated in TRAF2-null fibroblasts. More importantly, LIGHT-induced NF-κB and JNK activation can be restored by ectopic expression of TRAF2 in TRAF2–/– cells. Interestingly, in contrast to TNF signaling, the activation of both NF-κB and JNK by LIGHT was normal in RIP–/– and TRAF5–/– cells. Taken together, our data demonstrate that TRAF2, an important effector molecule in TNF signaling, plays a critical, non-redundant role in LIGHT-LTβR signaling. Our study documents that LIGHT is a potent activator of the MAP kinase JNK pathway. As a key modulator of the transcription factor AP-1, JNK may play a critical role in LIGHT-mediated cellular responses, in particular those involved in the development and maintenance of the lymphoid tissues. Although our current work has made progress in understanding mechanisms of LIGHT/LTβR signaling, much remains to be done. For instance, it will be important to determine how NF-κB and JNK pathways are specifically regulated by TRAF2 in response to LIGHT treatment and to discern the respective role of these two pathways in LIGHT-mediated cellular responses. Meeting these challenges will help us to better understand the physiological functions of LIGHT and LTβR.
The Role of SMURF1 in Controlling MEKK2 Degradation and Bone Homeostasis
Bone morphogenetic proteins (BMPs) and transforming growth factor-β (TGF-β) are important extracellular cues affecting skeleton biology. Through the action of a complex of membrane-bound type I and type II receptors, which possess a cytoplasmic serine-threonine kinase domain, BMP and TGF-β ligands activate a class of downstream signaling mediators, the SMAD proteins, to control target gene expression in the nucleus (Figure 1). Besides being phosphorylated by the receptors, SMAD proteins are regulated by other kinases and are subject to control by SMURF-mediated ubiquitin-proteasome degradation. We sought to understand the physiological functions of SMURF1, a member of the HECT family of ubiquitin E3 ligases. Figure 1. A) Villanueva Goldner staining of undecalcified tibia sections of mice at age 9 months. Scale bar, 0.4 mm; C, cortical bone; T, trabecular bone. B) Bone mineral density (BMD) measurements in 20 proximal-to-distal femoral divisions from mice at ages 1, 4, 9 and 14 months (M); WT, wild-type; *P < .05; ** P = .01. C) Regulation of MEKK2 degradation by SMURF1 in the context of bone morphogenetic protein (BMP) signaling and the osteogenic response. By augmenting MEKK2-JNK-JUN/ATF activity, the transcription of many genes involved in the osteogenic response (e.g., the gene for osteocalcin) can be sensitized to the BMP/SMADs, thereby providing an explanation for the phenotype observed in the SMURF1-deficient mice. Several reports, including an earlier one from my group, have suggested that SMURF1 plays a role in regulating osteoblast activity and thus bone formation. Initially, SMURF1 was shown as an E3 ligase for BMP pathway–specific SMAD1 and SMAD5; injecting Smurf1 cDNA into developing Xenopus embryos inhibited the expression of BMP-induced mesodermal markers (Zhu H et al. Nature 400: 687–93, 1999). We showed that SMURF1 can block BMP-2–induced osteogenic differentiation when introduced into pluripotent mouse C2C12 cells, which possess the potential to differentiate along either a myogenic or osteogenic lineage (Ying SX et al. J Biol Chem 278: 39029–36, 2003). Other groups demonstrated that SMURF1 also possesses the ability to target (1) Runx2, an osteoblast-specific transcription factor (Zhao M et al. J Biol Chem 278: 27939–44, 2003), (2) RhoA in cell protrusions (Wang HR et al. Science 302: 1775–9, 2003), and (3) the TGF-β type I receptor to proteasome degradation, but the receptor degradation requires interaction with SMAD7 (Ebisawa T et al. J Biol Chem 276: 12477–80, 2001). Thus, from these diverse arrays of biochemical activities, one can infer that SMURF1 has multiple tissue-specific functions in vivo. To evaluate SMURF1’s function in vivo, we inactivated the mouse Smurf1 allele through homologous recombination. The Smurf1–/– pups were born healthy with a fully developed skeleton but exhibited a gradual increase in bone mass as they aged (Figure 1). Smurf1–/– mice had thicker and denser bones, as most prominently shown in the cortical regions of the long bones. The cause of this age-dependent increase was traced to enhanced activities of osteoblasts, which became sensitized to BMP in the absence of SMURF1. Paradoxically, the phenotypic change in bone was not accompanied by a quantitative alteration in either SMAD proteins or TGF-β/BMP type I receptors, as would be expected from the known biochemical activities of SMURF1 in the TGF-β/BMP pathways. Instead, we found that a phosphorylated form of MEKK2, a MAP kinase kinase kinase (MAPKKK) upstream of JNK, was upregulated. Adding MEKK2 as well as activated JNK to either wild-type or SMURF1-deficient mice increased the activity of their osteoblasts, whereas adding a kinase activity–deficient mutant form of MEKK2 blocked their activity. We further showed that MEKK2 was ubiquitinated in normal but not SMURF1-deficient osteoblasts and that phosphorylated MEKK2 can be ubiquitinated with recombinant SMURF1 and other purified components of the ubiquitin system, demonstrating that MEKK2 is a bona fide physiological substrate of SMURF1. Although surprising, the lack of an observable change in SMAD levels or activities in Smurf1–/– mice is not without explanation. Mammalian genomes, murine included, contain two highly homologous SMURF genes: Smurf1 and Smurf2, the products of which are 80% identical in their amino acid sequences. Since Smurf1 and Smurf2 transcripts are co-expressed in osteoblasts and elsewhere, this raised the possibility of a compensatory action by SMURF2 in lieu of SMURF1, which sustained the seemingly normal development of Smurf1–/– embryos. Indeed, my group reported that SMURF2 also possesses the in vitro biochemical activity of targeting BMP-specific SMAD1 for proteasomal-mediated degradation (Zhang Y et al. Proc Natl Acad Sci U S A 98: 974–9, 2001). It is possible that there are non-overlapping requirements for the SMURF-mediated ubiquitination of different substrates, making the functions of SMURF1 and SMURF2 interchangeable in some instances. In the United States today, 10 million individuals suffer from osteoporosis, a debilitating disease of low bone mass and bone tissue deterioration, and 18 million more are at risk. Currently approved treatments mainly focus on the passive prevention of bone loss through nutritional supplements and inhibiting bone-resorbing osteoclast activity. As removal of SMURF1 leads to heightened bone growth without affecting the anatomic architecture of the bone, it may be desirable to develop a SMURF1 inhibitor for treating osteoporosis by promoting active bone growth.
Myelodysplastic Syndrome in Man and Mouse
Progress in understanding and treating MDS has been hampered by a lack of suitable animal models for this disease, as current models either do not transform to acute leukemia or develop an incompletely penetrant, myeloproliferative disease (MPD) with dysplastic features. To generate a mouse model for MDS, we took advantage of the observation that a large number of chromosomal translocations involving the NUP98 gene have been associated with MDS. In particular, NUP98 is fused to at least seven different HOX genes in patients with MDS. To generate a mouse model, we cloned a NUP98-HOXD13 (NHD13) fusion gene from a pediatric patient with MDS and inserted this fusion gene, under the control of Vav regulatory elements, into the mouse germline. Vav regulatory elements were used because they are known to drive expression in all hematopoietic cells. Clinically healthy NHD13 transgenic mice developed MDS that resembled the human disease in terms of peripheral blood cytopenias, dysplasia, and increased apoptosis in the context of a hypercellular or normocellular bone marrow (Figure 1). Furthermore, the clinical course of the disease was similar to that of human MDS; some mice remained overtly healthy for an extended observation period, with mild-to-moderate cytopenias and dysplasia. Other mice died of severe anemia, and still other mice died following transformation of MDS to acute leukemia. Figure 1. Dysplasia and apoptosis of hematopoietic cells from NHD13 transgenic mice. A) hypersegmented neutrophil, B) multinucleated erythroblast, C) giant platelet, D) lane 1, bone marrow from NHD13 mouse; lane 2, bone marrow from normal littermate; note oligonucleosomal “ladder” in lane 1. Following leukemic transformation, the NHD13 transgenic mice displayed a wide variety of distinct leukemic subtypes, including myeloid, erythroid, megakaryocytic, undifferentiated, pre-T lymphoblastic, and pre-B lymphoblastic. The frequent transformation of MDS to pre-T lymphoblastic and pre-B lymphoblastic leukemia in these mice was unanticipated because human MDS only rarely transforms into a lymphoid malignancy. However, other NUP98 fusion genes, such as NUP98-RAP1GDS1 and NUP98-ADD3, have been found in patients with pre-T lymphoblastic leukemia, suggesting that expression of NUP98 fusion genes can lead to T-cell as well as myeloid malignancies. It seems likely that the NHD13 transgene exerts its oncogenic effect through the inhibition of normal hematopoietic differentiation. This assertion is supported by experiments demonstrating that overexpression of NHD13 can inhibit megakaryocytic differentiation, and experiments showing that NHD13 embryonic stem (ES) cells are severely impaired in their ability to differentiate in vitro. In addition, these findings are consistent with reports showing that overexpression of HOX or NUP98-HOX fusion genes can impair differentiation in other systems. This is the first animal model for MDS that accurately recapitulates all of the key features of the human disease, including ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and progression to acute leukemia. Predictably, generation of this model has led to more questions than it has answered. First, we presume that additional, collaborating mutations occur as the disease evolves from MDS to acute leukemia. What are these mutations? To answer this question, we have enlisted the support of expert collaborators both within and outside the CCR. The techniques we are using include retroviral tagging, comparative genomic hybridization, and restriction landmark genome scanning (RLGS) to identify collaborating mutations; the retroviral tagging and RLGS approaches have both yielded promising leads. A second fundamental question is whether MDS, which can be regarded as a premalignant condition, can be transplanted. Again, we have enlisted the support of CCR investigators to help us answer this question, through the transplantation of NHD13 bone marrow into non-transgenic, syngeneic mice. If the disease is transplantable, can we identify an “MDS stem cell” analogous to the leukemic stem cells that have been described? Finally, new chemotherapy agents are often screened for efficacy using a panel of cell lines. There are no MDS cell lines that can be used for this type of experiment, however, because MDS cells do not grow well in vitro. The “MDS” cell lines that have been established were derived from patients in whom MDS had transformed to acute leukemia, and are therefore actually leukemic cell lines rather than MDS cell lines. To determine whether the NHD13 mice are a valid preclinical model for MDS, we are treating the mice with agents known to be effective in human patients with MDS, as a proof-of-concept experiment.
Efficient RNA Interference Delivery via Simian Virus 40 Vectors Packaged In Vitro
SV40 vectors packaged in vitro (SV40-IVPs, also called SV40 pseudovirions) are known to be efficient delivery systems for supercoiled DNA plasmids. SV40-IVPs can be used to efficiently transfer DNA up to 17.7 kb in size into a variety of mammalian cells both in vitro and in vivo. They are probably safer than the recombinant SV40 delivery system (Kimchi-Sarfaty C et al. Biotechniques 37: 270–5, 2004) or any other viral delivery system (Gottesman MM. Cancer Gene Ther 10: 501–8, 2003), because no packaging cell line or viral DNA is present. We demonstrated that the packaging and highly efficient delivery of siRNAs into two human cell lines, HeLa and human lymphoblastoids, in vitro can also be accomplished using SV40-IVPs. Our results were confirmed by way of a complete knockout of green fluorescent protein (GFP) expression. RNAi is mediated through the RNA-induced silencing complex, which has single-stranded RNA species 21 to 23 nucleotides long. This complex recognizes target RNAs and cleaves them in a position-dependent manner. Since some of this activity takes place within the cytoplasm, SV40-IVPs may serve as good carriers for such an agent. We used SV40-IVPs to deliver both plasmids expressing short hairpin RNAs (shRNAs) and synthetic siRNAs into human lymphoblastoid cells in suspension and HeLa adherent cells. Although delivery of synthetic siRNAs to adherent cells has previously been accomplished via polyplex delivery systems and cationic lipids, these methods do not work well on cells in suspension. Initially, we showed that SV40-IVPs can deliver shRNAs that interfere with GFP expression (SV40-IVP-shGFPs). Human lymphoblastoid cells that were transduced with a half reaction of SV40-IVP-shGFPs and a half reaction of SV40-IVP-GFPs did not show GFP expression (as transduced mock cells), in comparison with cells transduced with SV40-IVP-GFP only. We later demonstrated, by fluorescence-activated cell-sorting (FACS) and by confocal microscopy, a highly fluorescent signal in cells transduced by SV40-IVPs carrying fluorescence-tagged siRNA (SV40-IVP-siRNA.3.Fl), showing that siRNA can in fact be packaged and delivered by pseudovirions. A visible inhibition of GFP expression in HeLa cells, which continuously express GFP (under the influence of selective agent G418), was achieved by transducing cells with GFP-targeted siRNA (SV40-IVP-siGFP). A dose response of the packaged siGFP revealed that 1 μg of siGFP was sufficient to silence the GFP in 5 × 105 cells if it was packaged by SV40-IVPs. Finally, we compared the SV40-IVP delivery method to a lipid-transfection system using the HeLa cells that express high levels of GFP (Figure 1). The majority of the cells transduced with SV40-IVP-siRNA (SV40-IVP-siGFP) ceased GFP expression (approximately a 2 log shift), while lipid-transfected siRNA cells showed only a minor shift from the high levels of GFP observed in the control. The inhibition lasted longer using the pseudovirions, up to 6 days after transduction. Figure 1. Expression of small interfering RNA (siRNA) against HeLa cells stably expressing green fluorescent protein (GFP), with the siRNA delivered by simian virus 40 vectors packaged in vitro (SV40-IVP-siGFPs) or by lipid transduction. The majority of SV40-IVP-siGFP cells ceased GFP production (green), while most of the cells transfected with siRNAs (siGFPs) via lipids showed much higher expression of GFP (blue). Cells were analyzed 3 days after transduction. SV40-IVPs have several advantages over other delivery methods: They do not carry viral DNA, and therefore, there is no possibility of contamination. They are capable of transducing both non-dividing cells and cells in suspension, and they carry longer DNA fragments than other delivery methods. In this study, we showed that SV40-IVPs can be used for RNAi with very high efficiency and for a relatively long period of time.
Scientific Advisory CommitteeIf you have scientific news of interest to the CCR research community, please contact one of the scientific advisors (below) responsible for your areas of research.
CCR Frontiers in Science—StaffCenter for Cancer Research Robert H. Wiltrout, PhD, Director Deputy Directors Douglas R. Lowy, MD Editorial Staff Sue Fox, BA/BSW, Senior Editor * Palladian Partners, Inc. FOR INTERNAL USE ONLY |
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NCI’s HIV/AIDS Research—An Excellent Investment
Vaccines and Immunotherapy
ost of the 206 bones that make up the human skeleton are formed on an embryonic cartilage template, which is populated during development by the bone-forming osteoblasts derived from surrounding mesenchymal tissues. In a process called bone remodeling, osteoblasts and their partners osteoclasts help regulate the amount of bone tissue in the body throughout life, with new bone being formed by the osteoblasts and old bone being resorbed by the osteoclasts. With the advancement of mouse genetics approaches, investigators have begun to unravel the molecular underpinnings that govern this complex process. 
NA interference (RNAi) is the process of introducing small interfering RNA molecules (siRNAs) into eukaryotic cells to downregulate gene expression. There are two methods of introducing siRNAs into the cytoplasm: One is by using molecules like liposomes that are impregnated with double-stranded RNAs, and the other involves the use of viral vectors, in which the siRNAs are precursor RNAs expressed by the vectors (Huppi K et al. Mol Cell 17: 1–10, 2005). In our study, we propose a new delivery system, involving simian virus 40 (SV40) vectors, that can be incorporated into both methods. 
