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The large majority of human immunodeficiency virus type 1 (HIV-1) markers of disease progression/severity previously identified have been associated with alterations in host genetic and immune responses, with few studies focused on viral genetic markers correlate with changes in disease severity. This study presents a cross-sectional/longitudinal study of HIV-1 single nucleotide polymorphisms (SNPs) contained within the viral promoter or long terminal repeat (LTR) in patients within the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. HIV-1 LTR SNPs were found to associate with the classical clinical disease parameters CD4+ T-cell count and log viral load. They were found in both defined and undefined transcription factor binding sites of the LTR. A novel SNP identified at position 108 in a known COUP (chicken ovalbumin upstream promoter)/AP1 transcription factor binding site was significantly correlated with binding phenotypes that are potentially the underlying cause of the associated clinical outcome (increase in viral load and decrease in CD4+ T-cell count).
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Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Sitios de Unión/genética , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Estudios Transversales , Femenino , Estudios de Asociación Genética/métodos , Infecciones por VIH/metabolismo , Duplicado del Terminal Largo de VIH/genética , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Factores de Transcripción/genética , Carga Viral/genéticaRESUMEN
The future of treating inherited and acquired genetic diseases will be defined by our ability to introduce transgenes into cells and restore normal physiology. Here we describe an autogenous transgene regulatory system (ARES), based on the bacterial lac repressor, and demonstrate its utility for controlling the expression of a transgene in bacteria, eukaryotic cells, and in the retina of mice. This ARES system is inducible by the small non-pharmacologic molecule, Isopropyl ß-D-1-thiogalactopyranoside (IPTG) that has no off-target effects in mammals. Following subretinal injection of an adeno-associated virus (AAV) vector encoding ARES, luciferase expression can be reversibly controlled in the murine retina by oral delivery of IPTG over three induction-repression cycles. The ability to induce transgene expression repeatedly via administration of an oral inducer in vivo, suggests that this type of regulatory system holds great promise for applications in human gene therapy.
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Expresión Génica , Terapia Genética , Activación Transcripcional/efectos de los fármacos , Administración Oral , Animales , Dependovirus/genética , Genes Reporteros , Células HEK293 , Humanos , Isopropil Tiogalactósido/administración & dosificación , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Retina/metabolismo , TransgenesRESUMEN
Genetic pleiotropy, the phenomenon by which mutations in the same gene result in markedly different disease phenotypes, has proven difficult to explain with traditional models of disease pathogenesis. We have developed a model of pleiotropic disease that explains, through the process of basal exon skipping, how different mutations in the same gene can differentially affect protein production, with the total amount of protein produced correlating with disease severity. Mutations in the centrosomal protein of 290 kDa (CEP290) gene are associated with a spectrum of phenotypically distinct human diseases (the ciliopathies). Molecular biologic examination of CEP290 transcript and protein expression in cells from patients carrying CEP290 mutations, measured by quantitative polymerase chain reaction and Western blotting, correlated with disease severity and corroborated our model. We show that basal exon skipping may be the mechanism underlying the disease pleiotropy caused by CEP290 mutations. Applying our model to a different disease gene, CC2D2A (coiled-coil and C2 domains-containing protein 2A), we found that the same correlations held true. Our model explains the phenotypic diversity of two different inherited ciliopathies and may establish a new model for the pathogenesis of other pleiotropic human diseases.
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Enfermedad/genética , Exones/genética , Pleiotropía Genética , Predisposición Genética a la Enfermedad/genética , Modelos Genéticos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , FenotipoRESUMEN
The adaptation of human immunodeficiency virus type-1 (HIV-1) to an array of physiologic niches is advantaged by the plasticity of the viral genome, encoded proteins, and promoter. CXCR4-utilizing (X4) viruses preferentially, but not universally, infect CD4+ T cells, generating high levels of virus within activated HIV-1-infected T cells that can be detected in regional lymph nodes and peripheral blood. By comparison, the CCR5-utilizing (R5) viruses have a greater preference for cells of the monocyte-macrophage lineage; however, while R5 viruses also display a propensity to enter and replicate in T cells, they infect a smaller percentage of CD4+ T cells in comparison to X4 viruses. Additionally, R5 viruses have been associated with viral transmission and CNS disease and are also more prevalent during HIV-1 disease. Specific adaptive changes associated with X4 and R5 viruses were identified in co-linear viral sequences beyond the Env-V3. The in silico position-specific scoring matrix (PSSM) algorithm was used to define distinct groups of X4 and R5 sequences based solely on sequences in Env-V3. Bioinformatic tools were used to identify genetic signatures involving specific protein domains or long terminal repeat (LTR) transcription factor sites within co-linear viral protein R (Vpr), trans-activator of transcription (Tat), or LTR sequences that were preferentially associated with X4 or R5 Env-V3 sequences. A number of differential amino acid and nucleotide changes were identified across the co-linear Vpr, Tat, and LTR sequences, suggesting the presence of specific genetic signatures that preferentially associate with X4 or R5 viruses. Investigation of the genetic relatedness between X4 and R5 viruses utilizing phylogenetic analyses of complete sequences could not be used to definitively and uniquely identify groups of R5 or X4 sequences; in contrast, differences in the genetic diversities between X4 and R5 were readily identified within these co-linear sequences in HIV-1-infected patients.
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VIH-1/genética , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Algoritmos , Línea Celular , Genes Virales , VIH-1/metabolismo , HumanosAsunto(s)
Vectores Genéticos/metabolismo , Lipoproteína Lipasa/metabolismo , Animales , Ensayos Clínicos como Asunto , Dependovirus/genética , Evaluación Preclínica de Medicamentos , Terapia Genética , Vectores Genéticos/genética , Humanos , Hiperlipoproteinemia Tipo I/metabolismo , Hiperlipoproteinemia Tipo I/patología , Hiperlipoproteinemia Tipo I/terapia , Lipoproteína Lipasa/deficiencia , Lipoproteína Lipasa/genéticaRESUMEN
One-time gene therapy resulted in multiyear visual improvement, and new approaches were used to evaluate effects on retinal structure.
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Ceguera/genética , Atrofia Óptica Hereditaria de Leber/genética , Ceguera/terapia , Humanos , Atrofia Óptica Hereditaria de Leber/terapiaRESUMEN
The long terminal repeat (LTR) regulates gene expression of HIV-1 by interacting with multiple host and viral factors. Cross-sectional studies in the pre-HAART era demonstrated that single nucleotide polymorphisms (SNPs) in peripheral blood-derived LTRs (a C-to-T change at position 3 of C/EBP site I (3T) and at position 5 of Sp site III (5T)) increased in frequency as disease severity increased. Additionally, the 3T variant correlated with HIV-1-associated dementia. LTR sequences derived by longitudinal sampling of peripheral blood from a single patient in the DrexelMed HIV/AIDS Genetic Analysis Cohort resulted in the detection of the 3T and 5T co-selected SNPs before the onset of neurologic impairment, demonstrating that these SNPs may be useful in predicting HIV-associated neurological complications. The relative fitness of the LTRs containing the 3T and/or 5T co-selected SNPs as they evolve in their native patient-derived LTR backbone structure demonstrated a spectrum of basal and Tat-mediated transcriptional activities using the IIIB-derived Tat and colinear Tat derived from the same molecular clone containing the 3T/5T LTR SNP. In silico predictions utilizing colinear envelope sequence suggested that the patient's virus evolved from an X4 to an R5 swarm prior to the development of neurological complications and more advanced HIV disease. These results suggest that the HIV-1 genomic swarm may evolve during the course of disease in response to selective pressures that lead to changes in prevalence of specific polymorphisms in the LTR, env, and/or tat that could predict the onset of neurological disease and result in alterations in viral function.