RESUMEN
Adeno-associated viral vectors (AAV) are unique in their ability to transduce a variety of both dividing and nondividing cells, with significantly lower risk of random genomic integration and with no known pathogenicity in humans, but their role in ex vivo regional gene therapy for bone repair has not been definitively established. The goal of this study was to test the ability of AAV vectors carrying the cDNA for BMP-2 to transduce human mesenchymal stem cells (MSCs), produce BMP-2, and induce osteogenesis in vitro as compared with lentiviral gene therapy with a two-step transcriptional amplification system lentiviral vector (LV-TSTA). To this end, we created two AAV vectors (serotypes 2 and 6) expressing the target transgene; eGFP or BMP-2. Transduction of human MSCs isolated from bone marrow (BMSCs) or adipose tissue (ASCs) with AAV2-eGFP and AAV6-eGFP led to low transduction efficiency (BMSCs: 3.57% and 8.82%, respectively, ASCs: 6.17 and 20.2%, respectively) and mean fluorescence intensity as seen with FACS analysis 7 days following transduction, even at MOIs as high as 106. In contrast, strong eGFP expression was detectable in all of the cell types post transduction with LV-TSTA-eGFP. Transduction with BMP-2 producing vectors led to minimal BMP-2 production in AAV-transduced cells 2 and 7 days following transduction. In addition, transduction of ASCs and BMSCs with AAV2-BMP-2 and AAV6-BMP-2 did not enhance their osteogenic potential as seen with an alizarin red assay. In contrast, the LV-TSTA-BMP-2-transduced cells were characterized by an abundant BMP-2 production and induction of the osteogenic phenotype in vitro (p < 0.001 vs. AAV2 and 6). Our results demonstrate that the AAV2 and AAV6 vectors cannot induce a significant transgene expression in human BMSCs and ASCs, even at MOIs as high as 106. The LV-TSTA vector is significantly superior in transducing human MSCs; thus this vector would be preferable when developing an ex vivo regional gene therapy strategy for clinical use in orthopedic surgery applications.
Asunto(s)
Células Madre Mesenquimatosas , Tejido Adiposo , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/genética , Transducción Genética , TransgenesRESUMEN
Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCE HIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body's immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.
Asunto(s)
VIH-1/inmunología , Latencia del Virus/inmunología , Latencia del Virus/fisiología , Animales , Antirretrovirales/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Modelos Animales de Enfermedad , Infecciones por VIH/virología , Seropositividad para VIH/tratamiento farmacológico , VIH-1/patogenicidad , Humanos , Ratones , Receptor de Muerte Celular Programada 1/inmunología , Receptores Inmunológicos/inmunología , Nucleasas de los Efectores Tipo Activadores de la Transcripción/inmunología , Activación Viral , Replicación ViralRESUMEN
Most studies of HIV latency focus on the peripheral population of resting memory T cells, but the brain also contains a distinct reservoir of HIV-infected cells in microglia, perivascular macrophages, and astrocytes. Studying HIV in the brain has been challenging, since live cells are difficult to recover from autopsy samples and primate models of SIV infection utilize viruses that are more myeloid-tropic than HIV due to the expression of Vpx. Development of a realistic small animal model would greatly advance studies of this important reservoir and permit definitive studies of HIV latency. When radiation or busulfan-conditioned, immune-deficient NSG mice are transplanted with human hematopoietic stem cells, human cells from the bone marrow enter the brain and differentiate to express microglia-specific markers. After infection with replication competent HIV, virus was detected in these bone marrow-derived human microglia. Studies of HIV latency in this model would be greatly enhanced by the development of compounds that can selectively reverse HIV latency in microglial cells. Our studies have identified members of the CoREST repression complex as key regulators of HIV latency in microglia in both rat and human microglial cell lines. The monoamine oxidase (MAO) and potential CoREST inhibitor, phenelzine, which is brain penetrant, was able to stimulate HIV production in human microglial cell lines and human glial cells recovered from the brains of HIV-infected humanized mice. The humanized mice we have developed therefore show great promise as a model system for the development of strategies aimed at defining and reducing the CNS reservoir.
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Complejo SIDA Demencia/tratamiento farmacológico , Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Microglía/efectos de los fármacos , Inhibidores de la Monoaminooxidasa/farmacología , Proteínas del Tejido Nervioso/genética , Fenelzina/farmacología , Proteínas Represoras/genética , Complejo SIDA Demencia/genética , Complejo SIDA Demencia/fisiopatología , Complejo SIDA Demencia/virología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/virología , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Encéfalo/virología , Busulfano/toxicidad , Diferenciación Celular , Proteínas Co-Represoras , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , VIH-1/patogenicidad , VIH-1/fisiología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Ratones , Ratones Transgénicos , Microglía/metabolismo , Microglía/virología , Proteínas del Tejido Nervioso/metabolismo , Ratas , Proteínas Represoras/metabolismo , Trasplante Heterólogo , Latencia del Virus/efectos de los fármacos , Latencia del Virus/genética , Irradiación Corporal TotalRESUMEN
Transmission of drug-resistant HIV has been postulated to be a threat to current first-line antiretroviral therapy (ART) regimens and the efficacy of several antiretroviral-based preexposure prophylaxis (PrEP) strategies being tested. Here we evaluated the effect of the common tenofovir (TFV) resistance mutation K65R on vaginal HIV transmission. Our results demonstrate that despite no overt loss of overall replication competence in vivo, this mutation results in significantly reduced mucosal transmission. When transmitted, the mutant virus eventually reverted to the wild type in 2 of 3 animals examined.
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Adenina/análogos & derivados , Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral , Infecciones por VIH/transmisión , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Mutación Missense , Organofosfonatos/farmacología , Adenina/farmacología , Animales , Modelos Animales de Enfermedad , Femenino , Genotipo , VIH-1/clasificación , VIH-1/genética , Humanos , Ratones , Membrana Mucosa/virología , Supresión Genética , Tenofovir , Vagina/virologíaRESUMEN
Currently, over 15% of new HIV infections occur in children. Breastfeeding is a major contributor to HIV infections in infants. This represents a major paradox in the field because in vitro, breast milk has been shown to have a strong inhibitory effect on HIV infectivity. However, this inhibitory effect has never been demonstrated in vivo. Here, we address this important paradox using the first humanized mouse model of oral HIV transmission. We established that reconstitution of the oral cavity and upper gastrointestinal (GI) tract of humanized bone marrow/liver/thymus (BLT) mice with human leukocytes, including the human cell types important for mucosal HIV transmission (i.e. dendritic cells, macrophages and CD4⺠T cells), renders them susceptible to oral transmission of cell-free and cell-associated HIV. Oral transmission of HIV resulted in systemic infection of lymphoid and non-lymphoid tissues that is characterized by the presence of HIV RNA in plasma and a gradual decline of CD4⺠T cells in peripheral blood. Consistent with infection of the oral cavity, we observed virus shedding into saliva. We then evaluated the role of human breast milk on oral HIV transmission. Our in vivo results demonstrate that breast milk has a strong inhibitory effect on oral transmission of both cell-free and cell-associated HIV. Finally, we evaluated the effect of antiretrovirals on oral transmission of HIV. Our results show that systemic antiretrovirals administered prior to exposure can efficiently prevent oral HIV transmission in BLT mice.
Asunto(s)
Antirretrovirales/farmacología , Infecciones por VIH/transmisión , VIH-1/efectos de los fármacos , Leche Humana , Boca/virología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Duodeno/citología , Duodeno/inmunología , Duodeno/virología , Esófago/citología , Esófago/inmunología , Esófago/virología , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Macrófagos/citología , Macrófagos/inmunología , Ratones , Leche Humana/virología , Boca/citología , Boca/inmunología , Estómago/citología , Estómago/inmunología , Estómago/virología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Here we demonstrate that a combination of tenofovir, emtricitabine, and raltegravir effectively suppresses peripheral and systemic HIV replication in humanized BLT mice. We also demonstrate that antiretroviral therapy (ART)-treated humanized BLT mice harbor latently infected resting human CD4+ T cells that can be induced ex vivo to produce HIV. We observed that the levels of infected resting human CD4+ T cells present in BLT mice are within the range of those observed circulating in patients undergoing suppressive ART. These results demonstrate the potential of humanized BLT mice as an attractive model for testing the in vivo efficacy of novel HIV eradication strategies.
Asunto(s)
Modelos Animales de Enfermedad , Infecciones por VIH/virología , VIH/fisiología , Latencia del Virus , Animales , Fármacos Anti-VIH , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , VIH/efectos de los fármacos , VIH/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Humanos , Ratones , Ratones SCID , Latencia del Virus/efectos de los fármacosRESUMEN
Germ-free (GF) mice, which are depleted of their resident microbiota, are the gold standard for exploring the role of the microbiome in health and disease; however, they are of limited value in the study of human-specific pathogens because they do not support their replication. Here, we develop GF mice systemically reconstituted with human immune cells and use them to evaluate the role of the resident microbiome in the acquisition, replication and pathogenesis of two human-specific pathogens, Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV). Comparison with conventional (CV) humanized mice showed that resident microbiota enhance the establishment of EBV infection and EBV-induced tumorigenesis and increase mucosal HIV acquisition and replication. HIV RNA levels were higher in plasma and tissues of CV humanized mice compared with GF humanized mice. The frequency of CCR5+ CD4+ T cells throughout the intestine was also higher in CV humanized mice, indicating that resident microbiota govern levels of HIV target cells. Thus, resident microbiota promote the acquisition and pathogenesis of two clinically relevant human-specific pathogens.
RESUMEN
We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.
Asunto(s)
Reacciones Antígeno-Anticuerpo/genética , Linfocitos B/inmunología , Edición Génica/métodos , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Sistemas CRISPR-Cas , Línea Celular , Citidina Desaminasa/metabolismo , Anticuerpos Anti-VIH/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
The recently completed HIV prevention trials network study 052 is a landmark collaboration demonstrating that HIV transmission in discordant couples can be dramatically reduced by treating the infected individual with antiretroviral therapy (ART). However, the cellular and virological events that occur in the female reproductive tract (FRT) during ART that result in such a drastic decrease in transmission were not studied and remain unknown. Here, we implemented an in vivo model of ART in BM/liver/thymus (BLT) humanized mice in order to better understand the ability of ART to prevent secondary HIV transmission. We demonstrated that the entire FRT of BLT mice is reconstituted with human CD4+ cells that are shed into cervicovaginal secretions (CVS). A high percentage of the CD4+ T cells in the FRT and CVS expressed CCR5 and therefore are potential HIV target cells. Infection with HIV increased the numbers of CD4+ and CD8+ T cells in CVS of BLT mice. Furthermore, HIV was present in CVS during infection. Finally, we evaluated the effect of ART on HIV levels in the FRT and CVS and demonstrated that ART can efficiently suppress cell-free HIV-RNA in CVS, despite residual levels of HIV-RNA+ cells in both the FRT and CVS.
Asunto(s)
Fármacos Anti-VIH/farmacología , Cuello del Útero/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Tenofovir/farmacología , Vagina/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Células Cultivadas , Cuello del Útero/inmunología , Cuello del Útero/virología , Técnicas de Cocultivo , Femenino , Infecciones por VIH/prevención & control , Infecciones por VIH/transmisión , VIH-1/efectos de los fármacos , Humanos , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Pruebas de Sensibilidad Microbiana , ARN Viral/sangre , Receptores CCR5/metabolismo , Vagina/inmunología , Vagina/virología , Replicación ViralRESUMEN
The ability to direct human telomerase reverse transcriptase (hTERT) expression through either genetic control or tunable regulatory factors would advance not only our understanding of the transcriptional regulation of this gene, but also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and ZF nucleases (ZFNs) to target sequences within the hTERT promoter and exon-1. We were able to identify several active ZFTFs that demonstrate a broadly tunable response when screened by a cell-based transcriptional reporter assay. Using the same DNA-binding domains, we generated ZFNs that were screened in combinatorial pairs in cell-based extrachromosomal single-strand annealing (SSA) assays and in gene-targeting assays using stably integrated constructs. Selected ZFN pairs were tested for the ability to induce sequence changes in a Cel1 assay and we observed frequencies of genomic modification up to 18.7% at the endogenous hTERT locus. These screening strategies have pinpointed several ZFN pairs that may be useful in gene editing of the hTERT locus. Our work provides a foundation for using engineered ZF proteins (ZFPs) for modulation of the hTERT locus.Molecular Therapy - Nucleic Acids (2013) 2, e87; doi:10.1038/mtna.2013.12; published online 23 April 2013.
RESUMEN
Rectal microbicides are being developed to prevent new HIV infections in both men and women. We focused our in vivo preclinical efficacy study on rectally-applied tenofovir. BLT humanized mice (n = 43) were rectally inoculated with either the primary isolate HIV-1JRCSF or the MSM-derived transmitted/founder (T/F) virus HIV-1THRO within 30 minutes following treatment with topical 1% tenofovir or vehicle. Under our experimental conditions, in the absence of drug treatment we observed 50% and 60% rectal transmission by HIV-1JRCSF and HIV-1THRO, respectively. Topical tenofovir reduced rectal transmission to 8% (1/12; log rank p = 0.03) for HIV-1JRCSF and 0% (0/6; log rank p = 0.02) for HIV-1THRO. This is the first demonstration that any human T/F HIV-1 rectally infects humanized mice and that transmission of the T/F virus can be efficiently blocked by rectally applied 1% tenofovir. These results obtained in BLT mice, along with recent ex vivo, Phase 1 trial and non-human primate reports, provide a critically important step forward in the development of tenofovir-based rectal microbicides.