ABSTRACT
BACKGROUND: The gene therapeutic Cal-1 comprises the anti-HIV agents: (i) sh5, a short hairpin RNA to CCR5 that down-regulates CCR5 expression and (ii) maC46 (C46), a peptide that inhibits viral fusion with the cell membrane. These constructs were assessed for inhibition of viral replication and selective cell expansion in a number of settings. METHODS: HIV replication, selective outgrowth and cell surface viral binding were analysed with a single cycle infection assay of six pseudotyped HIV strains and a static and longitudinal passaging of MOLT4/CCR5 cells with HIV. Pronase digestion of surface virus and fluorescence microscopy assessed interactions between HIV virions and transduced cells. RESULTS: Cal-1 reduced CCR5 expression in peripheral blood mononuclear cells to CCR5Δ32 heterozygote levels. Even low level transduction resulted in significant preferential expansion in MOLT4/CCR5 gene-containing cells over a 3-week HIV challenge regardless of viral suppression [12.5% to 47.0% (C46), 46.7% (sh5), 62.2% (Dual), respectively]. The sh5 and Dual constructs at > 95% transduction also significantly suppressed virus to day 12 in the passage assay and all constructs, at varying percentage transduction inhibited virus in static culture. No escape mutations were present through 9 weeks of challenge. The Dual construct significantly suppressed infection by a panel of CCR5-using viruses, with its efficacy being independently determined from the single constructs. Dual and sh5 inhibited virion internalisation, as determined via pronase digestion of surface bound virus, by 70% compared to 13% for C46. CONCLUSIONS: The use of two anti-HIV genes allows optimal preferential survival and inhibition of HIV replication, with the impact on viral load being dependent on the percentage of gene marked cells.
Subject(s)
Genetic Therapy , HIV Infections/therapy , Receptors, CCR5/genetics , Recombinant Fusion Proteins/genetics , Gene Expression Regulation/genetics , HIV Infections/genetics , HIV Infections/virology , HIV-1/genetics , HIV-1/pathogenicity , Humans , Leukocytes, Mononuclear/virology , RNA, Small Interfering/genetics , RNA, Small Interfering/therapeutic use , Recombinant Fusion Proteins/therapeutic use , Transduction, Genetic , Viral Load/genetics , Virus Replication/geneticsABSTRACT
UNLABELLED: Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (<15%) occur in this region. To resolve this apparent discrepancy, we created a high-resolution genome-wide integration map of more than one million integration sites from CD34(+) hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form â¼60,000 tight clusters. These clusters comprise â¼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE: We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors.
Subject(s)
Enhancer Elements, Genetic , Genetic Vectors/physiology , Leukemia Virus, Murine/physiology , Virus Integration , Animals , Cells, Cultured , Genetic Therapy , Genetic Vectors/genetics , Genome, Human , Hematopoietic Stem Cells/virology , Histones/genetics , Histones/metabolism , Humans , Leukemia Virus, Murine/genetics , Mice , Mutagenesis, InsertionalABSTRACT
Gene therapy represents an alternative and promising anti-HIV modality to highly active antiretroviral therapy. It involves the introduction of a protective gene into a cell, thereby conferring protection against HIV. While clinical trials to date have delivered gene therapy to CD4+T cells or to CD34+ hematopoietic stem cells (HSC), the relative benefits of each of these two cellular targets have not been conclusively determined. In the present analysis, we investigated the relative merits of delivering a dual construct (CCR5 entry inhibitor + C46 fusion inhibitor) to either CD4+T cells or to CD34+ HSC. Using mathematical modelling, we determined the impact of each scenario in terms of total CD4+T cell counts over a 10 year period, and also in terms of inhibition of CCR5 and CXCR4 tropic virus. Our modelling determined that therapy delivery to CD34+ HSC generally resulted in better outcomes than delivery to CD4+T cells. An early one-off therapy delivery to CD34+ HSC, assuming that 20% of CD34+ HSC in the bone marrow were gene-modified (G+), resulted in total CD4+T cell counts ≥ 180 cells/ µL in peripheral blood after 10 years. If the uninfected G+ CD4+T cells (in addition to exhibiting lower likelihood of becoming productively infected) also exhibited reduced levels of bystander apoptosis (92.5% reduction) over non gene-modified (G-) CD4+T cells, then total CD4+T cell counts of ≥ 350 cells/ µL were observed after 10 years, even if initially only 10% of CD34+ HSC in the bone marrow received the protective gene. Taken together our results indicate that: 1.) therapy delivery to CD34+ HSC will result in better outcomes than delivery to CD4+T cells, and 2.) a greater impact of gene therapy will be observed if G+ CD4+T cells exhibit reduced levels of bystander apoptosis over G- CD4+T cells.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Computer Simulation , Genetic Therapy , HIV Infections , Hematopoietic Stem Cells/immunology , Models, Biological , Antigens, CD34 , Computational Biology , Disease Progression , Gene Transfer Techniques , HIV Fusion Inhibitors , HIV Infections/therapy , HIV Infections/virology , HIV-1/genetics , Humans , Receptors, CCR5 , Recombinant Fusion Proteins/geneticsABSTRACT
Analysis and mathematical modeling of T-lymphocyte perturbation following administration of granulocyte colony stimulating factor (G-CSF) and two large-scale aphereses are reported. 74 HIV-1 positive antiretroviral-treated individuals were infused with gene- or sham-transduced CD34+ hematopoietic stem cells (HSC) in a Phase II clinical trial. T cell numbers were examined in four phases: 1) during steady state; 2) increases in peripheral blood (PB) following G-CSF administration; 3) depletion post-aphereses and 4) reconstitution post HSC infusion. The present analysis provides the first direct estimate of CD4+ T cell distribution and trafficking in HIV-infected individuals on stable HAART, indicating that CD4+ T lymphocytes in PB represent 5.5% of the pool of CD4+ T lymphocytes that traffic to PB.
Subject(s)
HIV Infections/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Blood Component Removal , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Death/immunology , HIV Infections/therapy , Hematopoietic Stem Cell Transplantation , Humans , Lymphocyte Activation/immunology , Lymphocyte Count , Models, Theoretical , Phenotype , Time FactorsABSTRACT
Genetically distinct variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged since the start of the COVID-19 pandemic. Over this period, we developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021. Our platform facilitated viral variant isolation, rapid resolution of variant fitness using nasopharyngeal swabs and ranking of evasion of neutralizing antibodies. In late 2021, variant of concern Omicron (B1.1.529) emerged. Using our platform, we detected and characterized SARS-CoV-2 VOC Omicron. We show that Omicron effectively evades neutralization antibodies and has a different entry route that is TMPRSS2-independent. Our low-cost platform is available to all and can detect all variants of SARS-CoV-2 studied so far, with the main limitation being that our platform still requires appropriate biocontainment.
Subject(s)
COVID-19 , SARS-CoV-2 , Australia , COVID-19/diagnosis , Humans , Pandemics , SARS-CoV-2/geneticsABSTRACT
BACKGROUND: Gene therapy has the potential to counter problems that still hamper standard HIV antiretroviral therapy, such as toxicity, patient adherence and the development of resistance. RNA interference can suppress HIV replication as a gene therapeutic via expressed short hairpin RNAs (shRNAs). It is now clear that multiple shRNAs will likely be required to suppress infection and prevent the emergence of resistant virus. RESULTS: We have developed the first biologically relevant stochastic model in which multiple shRNAs are introduced into CD34+ hematopoietic stem cells. This model has been used to track the production of gene-containing CD4+ T cells, the degree of HIV infection, and the development of HIV resistance in lymphoid tissue for 13 years. In this model, we found that at least four active shRNAs were required to suppress HIV infection/replication effectively and prevent the development of resistance. The inhibition of incoming virus was shown to be critical for effective treatment. The low potential for resistance development that we found is largely due to a pool of replicating wild-type HIV that is maintained in non-gene containing CD4+ T cells. This wild-type HIV effectively out-competes emerging viral strains, maintaining the viral status quo. CONCLUSIONS: The presence of a group of cells that lack the gene therapeutic and is available for infection by wild-type virus appears to mitigate the development of resistance observed with systemic antiretroviral therapy.
Subject(s)
Genetic Therapy , HIV Infections/therapy , HIV-1/genetics , Models, Biological , RNA, Small Interfering/genetics , Anti-Retroviral Agents/therapeutic use , Drug Resistance, Viral , HIV Infections/drug therapy , Hematopoietic Stem Cells/immunology , Humans , Stochastic Processes , Virus Replication/geneticsABSTRACT
The HIV latent reservoir represents the major challenge to cure development. Residing in resting CD4+ T cells and myeloid cells at multiple locations in the body, including sanctuary sites such as the brain, the latent reservoir is not eliminated by ART and has the ability to reactivate virus replication to pre-therapy levels when ART is ceased. There are four broad areas of HIV cure research. The only successful cure strategy, thus far, is stem cell transplantation using naturally HIV resistant CCR5Δ32 stem cells. A second potential cure approach uses gene editing technology, such as zinc-finger nucleases and CRISPR/Cas9. Another two cure strategies aim to control the HIV reservoir, with polar opposite concepts; The "shock and kill" approach, which aims to "shock" or reactivate the latent virus and then "kill" infected cells via targeted immune responses. Lastly, the "block and lock" approach, which aims to enhance the latent virus state by "blocking" HIV transcription and "locking" the HIV promoter in a deep latent state via epigenetic modifications. "Shock and kill" approaches are a major focus of cure studies, however we predict that the increased specificity of "block and lock" approaches will be required for the successful development of a sustained HIV clinical remission in the absence of ART. This review focuses on the current research of novel "block and lock" approaches being explored to generate an HIV cure via induction of epigenetic silencing. We will also discuss potential future therapeutic delivery and the challenges associated with progressing "block and lock" cure approaches as these move toward clinical trials.
Subject(s)
HIV Infections , HIV-1 , CD4-Positive T-Lymphocytes , HIV Infections/drug therapy , HIV-1/genetics , Humans , Myeloid Cells , Virus Latency , Virus ReplicationABSTRACT
Gene silencing induced by RNAi represents a promising antiviral development strategy. This review will summarise the current state of RNAi therapeutics for treating acute and chronic human virus infections. The gene silencing pathways exploited by RNAi therapeutics will be described and include both classic RNAi, inducing cytoplasmic mRNA degradation post-transcription and novel RNAi, mediating epigenetic modifications at the transcription level in the nucleus. Finally, the challenge of delivering gene modifications via RNAi will be discussed, along with the unique characteristics of respiratory versus systemic administration routes to highlight recent advances and future potential of RNAi antiviral treatment strategies.
Subject(s)
RNAi Therapeutics , Virus Diseases/therapy , Acute Disease , Animals , Chronic Disease , Humans , RNA InterferenceABSTRACT
BACKGROUND: Gene therapy represents a new treatment paradigm for HIV that is potentially delivered by a safe, once-only therapeutic intervention. METHODS: Using mathematical modelling, we assessed the possible impact of autologous haematopoietic stem cell (HSC) delivered, anti-HIV gene therapy. The therapy comprises a ribozyme construct (OZ1) directed to a conserved region of HIV-1 delivered by transduced HSC (OZ1+HSC). OZ1+HSC contributes to the CD4+ T lymphocyte and monocyte/macrophage cell pools that preferentially expand under the selective pressure of HIV infection. The model was used to predict the efficacy of OZ1 in a highly active antiretroviral therapy (HAART) naïve individual and a HAART-experienced individual undergoing two structured treatment operations. In the standard scenario, OZ1+HSC was taken as 20% of total body HSC. RESULTS: For a HAART-naïve individual, modelling predicts a reduction of HIV RNA at 1 and 2 years post-OZ1 therapy of 0.5 log(10) and 1 log(10), respectively. Eight years after OZ1 therapy, the CD4+ T-lymphocyte count was 271 cells/mm(3) compared to 96 cells/mm(3) for an untreated individual. In a HAART-experienced individual HIV RNA was reduced by 0.34 log(10) and 0.86 log(10) at 1 and 2 years. The OZ1 effect was maximal when both CD4+ T lymphocytes and monocytes/macrophages were protected from successful, productive infection by OZ1. CONCLUSIONS: The modelling indicates a single infusion of HSC cell-delivered gene therapy can impact on HIV viral load and CD4 T-lymphocyte count. Given that gene therapy avoids the complications associated with HAART, there is significant potential for this approach in the treatment of HIV.
Subject(s)
Genetic Therapy , HIV Infections/drug therapy , HIV-1/drug effects , Hematopoietic Stem Cells , Models, Theoretical , RNA, Catalytic/genetics , Adult , Anti-HIV Agents/therapeutic use , Antiretroviral Therapy, Highly Active , CD4-Positive T-Lymphocytes/virology , Clinical Trials, Phase II as Topic , Computer Simulation , Drug Carriers , Humans , Macrophages/immunology , Macrophages/metabolism , RNA, Catalytic/therapeutic use , RNA, Viral/genetics , Treatment Outcome , Viral LoadABSTRACT
Ex vivo expansion of cord blood cells generally results in reduced stem cell activity in vivo. Glycogen synthase kinase-3beta (GSK-3beta) regulates the degradation of beta-catenin, a critical regulator of hematopoietic stem cells (HSCs). Here we show that GSK-3beta inhibition activates beta-catenin in cord blood CD34(+) cells and upregulates beta-catenin transcriptional targets c-myc and HoxB4, both known to regulate HSC self-renewal. GSK-3beta inhibition resulted in delayed ex vivo expansion of CD34(+) cells, yet enhanced the preservation of stem cell activity as tested in long-term culture with bone marrow stroma. Delayed cell cycling, reduced apoptosis, and increased adherence of hematopoietic progenitor cells to bone marrow stroma were observed in these long-term cultures treated with GSK-3beta inhibitor. This improved adherence to stroma was mediated via upregulation of CXCR4. In addition, GSK-3beta inhibition preserved severe combined immunodeficiency (SCID) repopulating cells as tested in the nonobese diabetic/SCID mouse model. Our data suggest the involvement of GSK-3beta inhibition in the preservation of HSC and their interaction with the bone marrow environment. Methods for the inhibition of GSK-3beta may be developed for clinical ex vivo expansion of HSC for transplantation. In addition, GSK-3beta inhibition suppressed leukemic cell growth via the induction of apoptosis mediated by the downregulation of survivin. Modulators of GSK-3beta may increase the range of novel drugs that specifically kill leukemic cells while sparing normal stem cells.
Subject(s)
Glycogen Synthase Kinase 3/antagonists & inhibitors , Hematopoietic Stem Cells/enzymology , Leukemia/enzymology , Leukemia/pathology , Animals , Antigens, CD34/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Coculture Techniques , Cord Blood Stem Cell Transplantation , Disease Models, Animal , Glycogen Synthase Kinase 3 beta , Hematopoietic Stem Cells/drug effects , Humans , Indoles/pharmacology , Mice , Mice, SCID , Oximes/pharmacology , Stromal Cells/drug effects , Stromal Cells/metabolism , Time Factors , Umbilical Cord/cytology , Umbilical Cord/drug effects , Umbilical Cord/enzymology , beta Catenin/metabolismABSTRACT
Interferon regulatory factor (IRF) 1 and its functional antagonist IRF2 were originally discovered as transcription factors that regulate the interferon-beta gene. Control of cell growth has led to the definition of IRF1 as a tumour suppressor gene and IRF2 as an oncogene. Clinically, approximately 70% of cases of acute myeloid leukaemia demonstrate dysregulated expression of IRF1 and/or IRF2. Our previous studies have shown that human leukaemic TF-1 cells exhibit abnormally high expression of both IRF1 and IRF2, the latter acting to abrogate IRF1 tumour suppression, making these cells ideal for analysis of down-regulation of IRF2 expression. A novel G418 screening protocol was developed and used for identifying effective siRNA that targets IRF2 (siIRF2). Using optimized siIRF2 in leukaemic TF-1 cells, IRF2 was down-regulated by approximately 70% at both mRNA and protein levels. Phenotypically, this resulted in growth inhibition associated with G2/M arrest as well as induction of polyploidy, differentiation and apoptosis. In contrast to these results, siIRF2 targeting did not affect normal haematopoietic stem/progenitor cell growth. These results indicate the potential utility of IRF2 inhibition as a therapeutic approach to cancer.
Subject(s)
Interferon Regulatory Factor-2/antagonists & inhibitors , Leukemia/therapy , RNA, Small Interfering/genetics , Antigens, CD34/analysis , Cell Cycle , Cell Line, Tumor , Hematopoiesis , Humans , Interferon Regulatory Factor-2/genetics , Leukemia/pathology , Lipopolysaccharide Receptors/analysisABSTRACT
OBJECTIVES: Mutations in ras oncogenes occur at high frequency in acute myeloid leukemia and myelodysplastic syndromes; however, the role of ras genes in leukemogenesis has not been clearly defined. Our previous studies have shown that expression of mutant N-ras (N-rasG13R, G to C transversion) in human hematopoietic progenitor cells (HPC) promotes myeloid differentiation and proliferation both in vitro and in a NOD/SCID mouse model. In the present study, we performed expression profiling to identify the transcriptome induced by N-rasG13R in human HPC, and analyzed the effect of mutant N-ras in sorted specific subpopulations of HPC. METHODS: cDNA microarray analysis was performed on cord blood CD34(+) cells transduced with a retrovirus containing GFP alone or in combination with mutant N-ras. Transduced cells were also sorted into factorial subpopulations according to CD34 and transgene expression, and analyzed in suspension or semi-solid methylcellulose culture. RESULTS: Among a variety of changes, including upregulation of cytokine genes, we found that N-rasG13R induced expression of the cyclin-dependent kinase inhibitors p16(INK4a) and p21(CIP1/WAF1). Analysis by RT-PCR revealed that increased p16(INK4a) and p21(CIP1/WAF1) occurred in the most primitive, CD34(+)/Ras(+) population but not in the more mature CD34(-)/Ras(+) cells or in the CD34(+)/Ras(-) cells. Moreover, N-rasG13R inhibited the proliferation of the primitive CD34(+)/Ras(+) cells, both in liquid culture and in colony assays. This growth suppression correlated with an increased proportion of myelomonocytic colonies and a decrease of erythroid colonies. In contrast, the growth of CD34(-)/Ras(+) cells and CD34(+)/Ras(-) HPC was not inhibited. CONCLUSIONS: These findings demonstrated the mutant N-ras induced transcriptome, and that this is associated with HPC growth suppression/myelomonocytic differentiation, and identify upregulation of cyclin inhibitors as key events in this process. The results indicate that ras mutation alone is not sufficient to induce leukemogenesis; collaborative secondary event(s) are involved in the process.
Subject(s)
Cell Differentiation/genetics , Cell Transformation, Neoplastic/genetics , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Genes, ras/genetics , Myeloid Progenitor Cells/metabolism , Up-Regulation/genetics , Animals , Antigens, CD34/biosynthesis , Cell Proliferation , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cytokines/biosynthesis , Cytokines/genetics , Gene Expression Regulation, Leukemic/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Mutation, Missense , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/pathology , Myeloid Progenitor Cells/pathologyABSTRACT
Activating mutations in ras oncogenes occur at high frequency in human malignancies and expression of activated ras in immortalized cells lines is generally transforming. However, somewhat paradoxically, ectopic expression of ras in some myeloid cell lines has been shown to induce growth suppression associated with up-regulation of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in a p16(INK4a), p15(INK4b), and p53 independent fashion. We have used cDNA array technology to compare the expression profile induced by activated N-ras (N-rasG13R) in growth-suppressed myeloid cells with that induced in myeloid cells, which are transformed by N-rasG13R. The expression profile induced in growth suppressed cells was consistent with differentiation and included the up-regulation of the transcription factor IFN regulatory factor-1 (IRF-1), a known transcriptional activator of p21(CIP/WAF1) expression and a target of oncogenic mutations associated with myeloid leukemia. Antisense suppression of IRF-1 prevented N-rasG13R-associated growth arrest and up-regulation of p21(CIP1/WAF1). These results define a novel tumor suppressive response to oncogenic signaling and provide a mechanistic link between growth suppression and differentiation in myeloid cells.
Subject(s)
DNA-Binding Proteins/physiology , Genes, ras/physiology , Myeloid Cells/physiology , Phosphoproteins/physiology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/genetics , Cell Growth Processes/physiology , Cyclin-Dependent Kinase Inhibitor p21 , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Disease Progression , Gene Expression Profiling , Humans , Interferon Regulatory Factor-1 , K562 Cells , Myeloid Cells/cytology , Oligonucleotide Array Sequence Analysis , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , U937 Cells , Up-RegulationABSTRACT
The efficient use of hematopoietic stem cells (HSC) for transplantation is often limited by the relatively low numbers of HSC collected. The ex vivo expansion of HSC for clinical use is a potentially valuable and safe approach to increase HSC numbers thereby increasing engraftment and reducing the risk of morbidity from infection. Here, we describe a protocol for the robust ex vivo expansion of human CD34(+) HSC isolated from umbilical cord blood. The protocol described can efficiently generate large numbers of HSC. We also describe a flow cytometry-based method using high-resolution division tracking to characterize the kinetics of HSC growth and differentiation. Utilizing the guidelines discussed, it is possible for investigators to use this protocol as presented or to modify it for their specific needs.
Subject(s)
Cell Culture Techniques/methods , Hematopoietic Stem Cell Transplantation/methods , Animals , Antigens, CD34/metabolism , Cell Differentiation/genetics , Cell Differentiation/physiology , Fetal Blood/cytology , Flow Cytometry , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/physiology , HumansABSTRACT
Activating mutations of the N-ras gene occur at relatively high frequency in acute myeloid leukemia and myelodysplastic syndrome. Somewhat paradoxically, ectopic expression of activated N-ras in primary hematopoietic cells and myeloid cell lines (in some cases) can lead to inhibition of proliferation. Expression of mutant N-ras in murine hematopoietic stem/progenitor cells is sufficient to induce myeloid malignancies, but these pathologies occur with long latency. This suggests that mutations that disable the growth suppressive properties of N-ras in hematopoietic cells are required for the development of frank malignancy. In the present work, the growth suppression induced by a mutant N-ras gene in U937 myeloid cells was used as the basis to screen a retroviral cDNA library for genes that prevent mutant N-ras-induced growth suppression (i.e., putative cooperating oncogenes). This screen identified the gene for the transcription factor interferon regulatory factor-2 (IRF-2), and as confirmation of the screen, overexpression of this gene in U937 cells was shown to inhibit mutant N-ras-induced growth suppression. Also recovered from the screen were two truncated clones of an uncharacterized gene (interim official symbol: PP2135). Overexpression of this truncated PP2135 gene in U937 cells did not appear to abrogate mutant N-ras-induced growth suppression, but rather appeared to confer an increased sensitivity of U937 cells to retroviral infection, accounting for the recovery of this gene from the genetic screen.
Subject(s)
Cell Proliferation , Genes, ras/physiology , Interferon Regulatory Factor-2/physiology , Mutation/genetics , Retroviridae/genetics , Blotting, Northern , Gene Expression , Gene Library , Humans , Polymerase Chain Reaction , U937 Cells/cytology , U937 Cells/metabolismABSTRACT
Acute myeloid leukaemia (AML) is the most common form of leukaemia in adults. Although of the order of 75-85% of patients will achieve complete remission after induction chemotherapy, long-term survival is still relatively low. Despite the progress in the rational design of drugs in disorders such as chronic myeloid leukaemia, AML lacks a single specific pathogenomic event to act as a drug target. Interferon regulatory factor 1 (IRF1) is a member of a family of related proteins that act as transcriptional activators or repressors. IRF1 and its functional antagonist IRF2 originally discovered as transcription factors regulating the interferon-beta (IFN-beta) gene, are involved in the regulation of normal haematopoiesis and leukaemogenesis. IRF1 appears to act as a tumour suppressor gene and IRF2 as an oncogene. IRF1 acts to repress IRF2 function through the repression of cyclin-dependent kinase (CDK) inhibitor p21WAF1 critical for cell growth control. It appears that the tumour suppression function of IRF1 is abolished by IRF2. This review focuses on the interaction between IRF1 and IRF2 in myeloid development and leukaemogenesis, particularly in relation to the Ras signalling pathway. IRF2 may be a viable and specific therapeutic target in human leukaemia.
Subject(s)
Interferon Regulatory Factor-1/physiology , Interferon Regulatory Factor-2/physiology , Leukemia, Myeloid/therapy , Acute Disease , Animals , Humans , Interferon Regulatory Factor-1/genetics , Interferon Regulatory Factor-2/genetics , Leukemia, Myeloid/genetics , Leukemia, Myeloid/metabolism , Mice , Models, Animal , Phenotype , Signal Transduction , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
HIV-1 infection requires the presence of the CD4 receptor on the target cell surface and a coreceptor, predominantly CC-chemokine receptor 5 (CCR5). It has been shown that individuals who are homozygous for a defective CCR5 gene are protected from HIV-1 infection. A novel self-inactivating lentiviral vector LVsh5/C46 (Cal-1) has been engineered to block HIV-1 infection with two viral entry inhibitors, conferring resistance to HIV-1 infection from both CCR5 and CXCR4 tropic strains. Cal-1 encodes a short hairpin RNA (sh5) to downregulate CCR5 and C46, an HIV-1 fusion inhibitor. Gene therapy by Cal-1 is aimed at transducing CD4+ T cells and CD34+ hematopoietic stem/progenitor cells in an autologous transplant setting. Pre-clinical safety and efficacy studies in vitro and in vivo (humanized mouse model and nonhuman primates) have shown that Cal-1 is safe with no indication of any toxicity risk and acts to decrease viral load and increase CD4 counts. Two clinical trials are underway using Cal-1: a phase I/II study to assess safety and feasibility in an adult HIV-1-positive population not on antiretroviral therapy (ART); and a second Fred Hutchinson Investigator Initiated phase I study to assess safety and feasibility in adults with HIV-1-associated non-Hodgkin or Hodgkin lymphoma.
Subject(s)
Genetic Therapy/methods , HIV Infections/therapy , HIV-1/physiology , Membrane Fusion , Receptors, CCR5 , Receptors, HIV/antagonists & inhibitors , Recombinant Fusion Proteins/biosynthesis , Adult , Animals , Biological Therapy/methods , CCR5 Receptor Antagonists , CD4 Lymphocyte Count , Clinical Trials as Topic , Down-Regulation , HIV Infections/genetics , HIV Infections/virology , HIV-1/genetics , Hematopoietic Stem Cells , Humans , Receptors, CCR5/biosynthesis , Receptors, CXCR4 , Recombinant Fusion Proteins/genetics , Viral LoadABSTRACT
While current antiretroviral therapy has significantly improved, challenges still remain in life-long targeting of HIV-1 reservoirs. Lentiviral gene therapy has the potential to deliver protective genes into the HIV-1 reservoir. However, inefficient reverse transcription (RT) occurs in HIV-1 reservoirs during lentiviral gene delivery. The viral protein Vpx is capable of increasing lentiviral RT by antagonizing the restriction factor SAMHD1. Incorporating Vpx into lentiviral vectors could substantially increase gene delivery into the HIV-1 reservoir. The feasibility of this Vpx approach was tested in resting cell models utilizing macrophages and dendritic cells. Our results showed Vpx exposure led to increased permissiveness of cells over a period that exceeded 2 weeks. Consequently, significant lower potency of HIV-1 antiretrovirals inhibiting RT and integration was observed. When Vpx was incorporated with anti-HIV-1 genes inhibiting either pre-RT or post-RT stages of the viral life-cycle, transduction levels significantly increased. However, a stronger antiviral effect was only observed with constructs that inhibit pre-RT stages of the viral life cycle. In conclusion this study demonstrates a way to overcome the major delivery obstacle of gene delivery into HIV-1 reservoir cell types. Importantly, incorporating Vpx with pre-RT anti-HIV-1 genes, demonstrated the greatest protection against HIV-1 infection.
ABSTRACT
HIV-1 infection has been transformed by combined anti-retroviral therapy (ART), changing a universally fatal infection into a controllable infection. However, major obstacles for an HIV-1 cure exist. The HIV latent reservoir, which exists in resting CD4+ T cells, is not impacted by ART, and can reactivate when ART is interrupted or ceased. Additionally, multi-drug resistance can arise. One alternate approach to conventional HIV-1 drug treatment that is being explored involves gene therapies utilizing RNA-directed gene regulation. Commonly known as RNA interference (RNAi), short interfering RNA (siRNA) induce gene silencing in conserved biological pathways, which require a high degree of sequence specificity. This review will provide an overview of the silencing pathways, the current RNAi technologies being developed for HIV-1 gene therapy, current clinical trials, and the challenges faced in progressing these treatments into clinical trials.
ABSTRACT
We have focused on gene therapy approaches to induce functional cure/remission of HIV-1 infection. Here, we evaluated the safety and efficacy of the clinical grade anti-HIV lentiviral vector, Cal-1, in pigtailed macaques (Macaca nemestrina). Cal-1 animals exhibit robust levels of gene marking in myeloid and lymphoid lineages without measurable adverse events, suggesting that Cal-1 transduction and autologous transplantation of hematopoietic stem cells are safe, and lead to long-term, multilineage engraftment following myeloablative conditioning. Ex vivo, CD4+ cells from transplanted animals undergo positive selection in the presence of simian/human immunodeficiency virus (SHIV). In vivo, Cal-1 gene-marked cells are evident in the peripheral blood and in HIV-relevant tissue sites such as the gastrointestinal tract. Positive selection for gene-marked cells is observed in blood and tissues following SHIV challenge, leading to maintenance of peripheral blood CD4+ T-cell counts in a normal range. Analysis of Cal-1 lentivirus integration sites confirms polyclonal engraftment of gene-marked cells. Following infection, a polyclonal, SHIV-resistant clonal repertoire is established. These findings offer strong preclinical evidence for safety and efficacy of Cal-1, present a new method for tracking protected cells over the course of virus-mediated selective pressure in vivo, and reveal previously unobserved dynamics of virus-dependent T-cell selection.