RESUMEN
Gene therapy is an experimental treatment modality under investigation for applications to HIV-1 infection. We have developed retroviral vectors carrying anti-HIV-1 genes, demonstrated that these genes cause significant suppression of HIV-1 replication in cultures of primary hematopoietic cells, and performed a clinical trial in pediatric AIDS patients. Four HIV-1-infected children and adolescents underwent bone marrow harvest from which CD34+ cells were isolated and transduced by a retroviral vector carrying an RRE decoy gene. The cells were re-infused into the subjects, without complications, showing that gene transfer in pediatric AIDS patients in safe and feasible. However, gene-containing leukocytes in the peripheral blood were seen only at a low level and only in the first months following cell infusion. To attain some degree of efficacy, it will be necessary to achieve a higher level of gene transfer and to obtain sustained gene expression. We are currently developing new gene transfer methods and vectors designed to improve the results in future trials. If it becomes possible to reach the ideal goal of producing high percentages of T lymphocytes and monocytic cells that are resistant to HIV-1 infection, gene therapy could serve as a complement to antiretroviral drug therapy and help to sustain immunologic function.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/terapia , Trasplante de Médula Ósea , Terapia Genética , Infecciones por VIH/terapia , VIH-1 , Adolescente , Niño , Ensayos Clínicos como Asunto , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , VIH-1/genética , VIH-1/fisiología , Humanos , Retroviridae , Replicación ViralRESUMEN
Genetic modification of hematopoietic stem cells with genes that inhibit replication of human immunodeficiency virus-1 (HIV-1) could lead to development of T lymphocytes and monocytic cells resistant to HIV-1 infection after transplantation. We performed a clinical trial to evaluate the safety and feasibility of this procedure, using bone marrow from four HIV-1-infected pediatric subjects (ages 8 to 17 years). We obtained bone marrow, isolated CD34(+) cells, performed in vitro transduction with a retroviral vector carrying a rev-responsive element (RRE) decoy gene, and reinfused the cells into these subjects with no evidence of adverse effects. The levels of gene-containing leukocytes in peripheral blood samples in the 1 year after gene transfer/cell infusion have been extremely low. These observations support the potential of performing gene therapy for HIV-1 using hematopoietic cells, but emphasize the need for improved gene transfer techniques.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/terapia , Genes rev , Terapia Genética , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Trasplante de Células Madre Hematopoyéticas , Replicación Viral/genética , Síndrome de Inmunodeficiencia Adquirida/genética , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/patología , Adolescente , Diferenciación Celular , Niño , Técnicas de Transferencia de Gen , Vectores Genéticos , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/fisiología , Humanos , Proyectos Piloto , Retroviridae , Linfocitos T/inmunologíaRESUMEN
Retroviral vectors based on the Moloney murine leukemia virus (Mo-MuLV) are among the most commonly used vectors for stable gene transfer into mammalian cells. However, expression from the transcription unit of the Mo-MuLV long terminal repeat (LTR) has often been unsatisfactory. Transcriptional suppression of retroviral vectors in vitro in embryonal carcinoma (EC) cells and in vivo in hematopoietic stem cells (HSCs) has been associated with increased levels of cytosine methylation in the vector 5' LTR. To obtain a comprehensive picture of the methylation pattern in the 5' LTR of retroviral vectors, we employed the bisulfite genomic sequencing technique, which allows detection of the methylation pattern of every CpG dinucleotide in a target sequence. We studied the 5' LTR within the Mo-MuLV-based vector, LN, and a series of multiply modified vectors, which show improved expression in vitro and in vivo. Methylation patterns of the vectors were compared in PA317 (3T3-derived) fibroblasts, which are permissive for expression from all of the vectors, and in F9 embryonal carcinoma (EC) cells, which are restrictive for expression from the parental Mo-MuLV LTR but show improved expression from the modified vectors. These analyses revealed that the levels of methylation of CpG dinucleotides were globally consistent throughout the entire LTR, including the region of transcriptional factor binding. All vectors showed no measurable methylation of CpG dinucleotides throughout the 5' LTR in the PA317 fibroblasts. The CpG dinucleotides of the standard Mo-MuLV-based vector (LN) were highly methylated in F9 EC cells (49.1%). The doubly modified vector, MD-neo, which did not show improved expression, exhibited a relatively high level of methylation (45%), similar to that found in the LN vector. In contrast, the CpG dinucleotides of the triply modified vectors, which showed improved expression in EC cells (MND-neo and MTD-neo), were much less methylated (26.2 and 23.4%, respectively). The results extend our previous findings of an inverse correlation between gene expression and methylation of cytosine residues of the LTR of retroviral vectors.
Asunto(s)
Citosina/análisis , Citosina/metabolismo , Retroviridae/genética , Análisis de Secuencia de ADN/métodos , Secuencias Repetidas Terminales , Animales , Northern Blotting , Carcinoma Embrionario/virología , Islas de CpG , Metilación de ADN , Cartilla de ADN , Regulación Viral de la Expresión Génica , Vectores Genéticos , Ratones , Virus de la Leucemia Murina de Moloney/genética , Sulfitos , Antígenos Thy-1/genética , Células Tumorales CultivadasRESUMEN
Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.
Asunto(s)
Adenosina Desaminasa/inmunología , Antígenos CD34/inmunología , Trasplante de Células Madre Hematopoyéticas , Linfocitos T/inmunología , Inmunología del Trasplante/inmunología , Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Animales , Animales Recién Nacidos , Línea Celular , Citometría de Flujo , Frecuencia de los Genes , Granulocitos/inmunología , Humanos , Leucocitos Mononucleares/inmunología , Recuento de Linfocitos , Ratones , Ratones SCID , Polietilenglicoles , Linfocitos T/efectos de los fármacos , Transformación Genética , Trasplante Autólogo , Cordón UmbilicalRESUMEN
Eight Nubian and eight Alpine dairy goat does were used in a crossover experimental design to determine the effect of 30 min of isolation on behavior and plasma concentrations of thyroxine (T4), triiodothyronine (T3), cortisol, norepinephrine (NOR), and epinephrine (EPI). Isolation was hypothesized to produce an emotional state analogous to fear. Focal animal behavior was recorded for the initial five min of isolation. Blood samples were obtained via jugular cannulae at 0 (immediately prior), 10, 20, 30 (during isolation), 40, 50 and 60 min (after return to their group). Response to isolation was characterized physiologically by increased plasma concentrations of NOR (p less than 0.01), but not T3, T4, cortisol or EPI, indicating a sympathetic discharge. Isolated goats also vocalized more frequently (p less than 0.01) and spent a greater amount of time sniffing, trotting and rearing (p less than 0.05). The Nubian does reacted more strongly (elevated NOR, trotting and rearing, p less than 0.01) to isolation than the Apline does.
Asunto(s)
Nivel de Alerta/fisiología , Conducta Animal/fisiología , Cabras/fisiología , Aislamiento Social , Animales , Epinefrina/sangre , Miedo/fisiología , Femenino , Hidrocortisona/sangre , Actividad Motora/fisiología , Norepinefrina/sangre , Medio Social , Tiroxina/sangre , Triyodotironina/sangreRESUMEN
Eight Nubian dairy goat does in one experiment, and eight Alpine dairy goat does in a second experiment, were randomly allotted to food-thwarted or fed groups in a crossover experimental design. Food thwarting was hypothesized to produce an emotional state analogous to frustration. After a 1-week training period during which the goats of both breeds were conditioned to being simultaneously fed in adjacent feeding stalls, frustration was induced in half the goats by feeding only alternate does. Focal animal behavior was recorded for the initial five min after feeding frustration commenced. Blood samples were collected via a jugular cannula before, during and after frustration was induced for thyroxine (T4), triiodothyronine (T3), cortisol, norepinephrine (NOR), and epinephrine (EPI) determinations. Food thwarting was characterized by increased plasma concentrations of NOR, and increased incidences of pawing, head movements, mouthing of objects, behaviors directed toward neighboring does being fed, and rearing (p less than 0.01). When data were pooled across experiments, breed had a strong influence on cortisol (p less than 0.05), with Nubian does having higher concentrations regardless of treatment. Concentrations of hormones were not significantly correlated with behaviors. These findings suggest that frustration may elicit a discharge of NOR but not EPI.
Asunto(s)
Nivel de Alerta/fisiología , Conducta Animal/fisiología , Privación de Alimentos/fisiología , Frustación , Cabras/fisiología , Animales , Epinefrina/sangre , Conducta Alimentaria/fisiología , Femenino , Hidrocortisona/sangre , Actividad Motora/fisiología , Norepinefrina/sangre , Tiroxina/sangre , Triyodotironina/sangreRESUMEN
Full-length infectious DNA clones were constructed for four distinct phenotypic variants of beet curly top virus (BCTV). Southern hybridization assays indicated that each cloned BCTV genome shared sequence homology with pBCT-028, a full-length infectious DNA clone of a California isolate of BCTV previously characterized by others. Restriction endonuclease maps of the cloned BCTV genomes were distinct from one another. Infectivity assays determined that plasmids containing tandem repeats of BCTV genomes were generally more infectious than excised linear DNA inserts. Progeny virus, derived from plants inoculated with cloned DNAs, differed in their ability to infect sugarbeet, Beta vulgaris L., and the severity of symptoms produced in B. vulgaris and other experimental hosts.
Asunto(s)
Virus de Plantas/genética , Southern Blotting , Clonación Molecular , ADN Viral/genética , Fenotipo , Virus de Plantas/patogenicidad , Plantas/microbiología , Mapeo Restrictivo , Replicación ViralRESUMEN
Previous studies have suggested that experimental alterations in adrenomedullary reactivity, i.e., changes in catecholamine release in response to a standard dose of acetylcholine, may be partially accounted for by changes occurring at the level of the adrenal medulla itself, independent of both the central nervous system and the innervation of the adrenal gland. The present study was designed to investigate the morphology of adrenal chromaffin cells in rats subjected to chronic hypoglycemia induced by long acting insulin, and to assess this morphology in terms of associated changes in catecholamine content and release. Surgically isolated, perfused adrenal gland preparations were utilized to characterize the functional release of catecholamines from the adrenal medulla. Pretreatment with long acting insulin resulted in a selective depletion of epinephrine stores and acetylcholine-mediated epinephrine release, but did not appear to significantly affect either the levels or the release of norepinephrine. The biochemical effects of long acting insulin persisted for several days after termination of the treatment, exhibiting a gradual recovery over a period of approximately 5 days. Electron microscopic examination of the adrenal chromaffin cells revealed a progressive degranulation and vacuolization of numerous chromaffin cells followed by a compensatory biosynthetic response and a gradual recovery toward the morphology of control cells. The functional release of catecholamines from adrenal chromaffin cells was further examined in preparations of perfused adrenal slices. Acetylcholine-mediated catecholamine release was significantly decreased in slices of adrenal glands prepared from insulin treated rats when compared with that of control animals.(ABSTRACT TRUNCATED AT 250 WORDS)