RESUMEN
Primate bone marrow cells were infected with a retroviral vector carrying the genes for human adenosine deaminase (h-ADA) and bacterial neomycin resistance (neor). The infected cells were infused back into the lethally irradiated donor animals. Several monkeys fully reconstituted and were shown to express the h-ADA and neor genes at low levels in their recirculating hematopoietic cells for short periods of time.
Asunto(s)
Adenosina Desaminasa/genética , Nucleósido Desaminasas/genética , Retroviridae/genética , Transformación Genética , Animales , Células de la Médula Ósea , Trasplante de Médula Ósea , ADN/genética , Resistencia a Medicamentos/genética , Células Madre Hematopoyéticas/enzimología , Humanos , Linfocitos/enzimología , Macaca fascicularis , Monocitos/enzimología , Neomicina , Hibridación de Ácido Nucleico , Irradiación Corporal TotalRESUMEN
Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and IL-2 appears to produce dramatic regressions in patients with metastatic melanoma and renal cancer. However, the in vivo mechanism of TIL function is not known. We conducted an UCLA Human Subject Protection Committee, Recombinant DNA Advisory Committee, and FDA-approved clinical trial using genetically-marked TIL to test the hypothesis that these cells have unique, tumor-specific in vivo trafficking patterns. TIL and PBL (as a control effector cell population) were isolated and expanded in parallel in vitro in IL-2-containing medium for 4-6 wk. During the expansion, TIL and PBL were separately transduced with the amphotropic retroviral vectors LNL6 and G1Na. Transduced TIL and PBL were coinfused into patients and their respective numbers measured in tumor, peripheral blood, and normal tissues; integrated provirus could be quantitated and distinguished by DNA PCR. Nine patients were treated (six melanoma, three renal) and received between 4.5 x 10(8) and 1.24 x 10(10) total cells. Both "marked" TIL and PBL could be detected circulating in the peripheral blood, in some patients for up to 99 d after infusion. Marked TIL and/or PBL could be detected in tumor biopsies in six of nine patients as early as day 6 and as late as day 99 after infusion. No convincing pattern of preferential trafficking of TIL vs. PBL to tumor was noted. Moreover, concurrent biopsies of muscle, fat, and skin demonstrated the presence of TIL/PBL in comparable or greater numbers than in tumor in five patients. The results of this double gene marking trial provide interesting insights into the life span and trafficking of adoptively transferred lymphocytes, but do not support the hypothesis that TIL specifically traffic to tumor deposits.
Asunto(s)
Linfocitos Infiltrantes de Tumor/citología , Adulto , Anciano , Secuencia de Bases , Carcinoma de Células Renales/terapia , Cartilla de ADN/química , Estudios de Evaluación como Asunto , Femenino , Marcadores Genéticos , Vectores Genéticos , Humanos , Inmunización Pasiva , Masculino , Melanoma/terapia , Persona de Mediana Edad , Datos de Secuencia MolecularRESUMEN
Disseminated neuroblastoma is a malignancy of children often treated by intensive chemotherapy/radiotherapy followed by autologous bone marrow transplantation (ABMT). A high proportion of those treated subsequently relapse. It is unknown if relapse is a consequence of residual disease in the patient or of contaminating malignant cells remaining in the infused marrow, which, of necessity, is harvested and stored prior to ablative chemotherapy/radiotherapy. The assumption that residual cells in the infused marrow contribute to relapse has lead to the adoption of marrow purging prior to reinfusion. However, neither the necessity nor the efficacy of the procedure have been established. We now show how retroviral-mediated gene transfer using the LNL6 vector may resolve this issue. Clonogenic neuroblastoma cells in patient marrow can be transduced and the NEOR gene detected by observing individual neuroblastoma cell colony growth in G418, and by polymerase chain reaction (PCR) of individual colonies. Efficiency of transduction is between 0 and 13.5%. If marrow is exposed to LNL6 prior to infusion and marked cells are detected at the time of relapse, this would demonstrate that infused marrow contributed to disease recurrence. The technique could then be used to analyze the efficacy of marrow purging techniques. Since normal progenitor cells from these patients are also marked, the technique can be used to study factors that modify reconstitution and transducibility of infused marrow. Clinical studies using this approach have now begun.
Asunto(s)
Trasplante de Médula Ósea , Recurrencia Local de Neoplasia , Neuroblastoma/patología , Retroviridae/genética , Transfección , Secuencia de Bases , ADN/análisis , Resistencia a Medicamentos/genética , Marcadores Genéticos , Gentamicinas/farmacología , Humanos , Datos de Secuencia Molecular , Neuroblastoma/genética , Neuroblastoma/cirugía , Fenotipo , Reacción en Cadena de la Polimerasa , Transducción Genética , Trasplante AutólogoRESUMEN
We investigated whether directly injecting retroviral vectors into preimmune fetuses could result in the transfer and long-term expression of exogenous genes. Twenty-nine preimmune sheep fetuses were injected with helper-free retroviral vector preparations. Twenty-two fetuses survived to term, 4 of which were sacrificed at birth. Of the remaining 18 animals, 3 were controls and 15 had received vector preparations. Twelve of these 15 animals demonstrated transduction of hematopoietic cells when blood and marrow were analyzed by neo(r)-specific PCR. Eight experimental sheep have been followed for 5 years, during which time we have consistently observed proviral DNA and G418-resistant hematopoetic progenitors. The G418-resistant colonies were positive when analyzed by neo(r)-specific PCR. neo(r) gene expression was also demonstrated using several immunological and biochemical methods. The transduction of hematopoietic stem cells was confirmed when lambs transplanted with bone marrow from in utero-transduced sheep exhibited neo(r) activity in marrow and blood. Vector distribution was widespread in primary animals without pathology. PCR analysis indicates that the germ line was not altered. These studies demonstrate that direct injection of an engineered retrovirus is a feasible means of safely delivering a foreign gene to a developing fetus and achieving long-term expression without modifying the germ line of the recipient.
Asunto(s)
Enfermedades Fetales/terapia , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Kanamicina Quinasa/genética , Kanamicina Quinasa/metabolismo , Retroviridae/genética , Animales , Southern Blotting , Ensayo de Inmunoadsorción Enzimática , Células Precursoras Eritroides/efectos de los fármacos , Femenino , Enfermedades Fetales/genética , Técnica del Anticuerpo Fluorescente , Técnicas de Transferencia de Gen , Gentamicinas/farmacología , Trasplante de Células Madre Hematopoyéticas , Hibridación Fluorescente in Situ , Inyecciones Intraperitoneales , Reacción en Cadena de la Polimerasa , Embarazo , Análisis de Secuencia de ADN , OvinosRESUMEN
The in vivo fate of amphotropic murine leukemia retrovirus was studied in five rhesus monkeys. Retrovirus infused intravenously into 3 normal animals and 1 immunosuppressed animal was cleared rapidly from the circulation and subsequent viremia has not been detected (mean follow-up of 27.4 months). A fifth monkey was immunosuppressed and transplanted with virus-producing autologous fibroblasts in addition to an intraperitoneal injection of virus. This animal was viremic for 2 days and its lymph node cells and peripheral blood mononuclear cells were shown to be producing virus for up to 22 days post-inoculation, but subsequently has been negative after 17.0 months of analysis. In the 5 animals studied (combined mean follow-up of 25.7 months), clinical illness has not been identified at any time. Therefore, murine amphotropic retroviruses do not appear to pose an acute health risk.
Asunto(s)
Virus de la Leucemia Murina/patogenicidad , Complejo Relacionado con el SIDA/microbiología , Animales , Antígenos Virales/sangre , Secuencia de Bases , Western Blotting , Fibroblastos/microbiología , Fibroblastos/trasplante , Terapia de Inmunosupresión , Inyecciones Intravenosas , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/inmunología , Macaca mulatta , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Replicación ViralRESUMEN
To evaluate whether marrow contributes to relapse after autologous bone marrow transplantation (AuBMT) for acute leukemia, transplanted marrow was marked with the G1N retroviral vector (Genetic Therapy Inc.) containing the neomycin phosphotransferase gene (neo). Between April 1992 and August 1993, 4 patients were transplanted for acute myeloid leukemia (AML) in second complete remission (CR) and 1 patient for acute lymphoid leukemia in first CR. An average of 12.4% (range 5-19%) of transplanted marrow mononuclear cells were exposed to G1N vector for 4 hr. In the vector-treated portion of the marrow, 4.9% of GM-CFU and 3.6% of erythroid burst-forming units (BFU-E) were resistant to G418 in vitro. In the 5 patients, the polymerase chain reaction (PCR) detected the neo sequence on only two occasions after AuBMT. Of 4 patients surviving 1 year after transplantation, only 1 had evidence of gene marked cells by PCR. Two AML patients have relapsed, one of whom had evidence of neo sequences in the bone marrow at day 100 but not at relapse 11 months after AuBMT. The second patient relapsed 18 months after AuBMT but never had PCR evidence of neo sequences before or after relapse. Our results indicate vector-transduced autologous bone marrow from heavily pretreated adults with acute leukemia mark with low efficiency, although vector sequences have been detected in bone marrow and peripheral blood up to 1 year after transplant. Of the 2 relapsed patients, no evidence of vector-marked leukemic blasts have been detected.
Asunto(s)
Trasplante de Médula Ósea , Técnicas de Transferencia de Gen , Vectores Genéticos , Leucemia Linfoide/terapia , Leucemia Mieloide/terapia , Retroviridae/genética , Enfermedad Aguda , Adulto , Seguridad de Productos para el Consumidor , Femenino , Estudios de Seguimiento , Células Madre Hematopoyéticas , Humanos , Masculino , Persona de Mediana Edad , Trasplante AutólogoRESUMEN
Replication-defective retroviral vectors were used for ex vivo gene transfer into rat liver grafts under conditions mimicking clinical liver transplantation. Supernatant containing single- and double-gene vectors encoding for either the human IL-7 and/or neomycin phosphotransferase genes were used to perfuse the liver grafts during cold ischemia before transplantation. Whole liver grafts were perfused with vector supernatant or medium only. Reduced-size liver grafts (50% hepatectomy) were similarly perfused either immediately after reduction or 24 hr later after induction of active hepatocyte division. After transplantation of these grafts in orthotopic position, the liver tissue was removed at specified intervals, and genomic DNA and mRNA were examined for proviral sequences and expression. Stable integration of the proviral sequences was detected only in reduced-size grafts transplanted 24 hr after hepatectomy. Proviral message of both neomycin phosphotransferase and human IL-7 were present up to 21 days after transduction. This study demonstrates efficient ex vivo gene transfer to donor liver grafts. Gene transfer to livers before transplantation carries the potential to modulate immunogenicity and alter the antigraft immune response.
Asunto(s)
Técnicas de Transferencia de Gen , Trasplante de Hígado/métodos , Animales , Expresión Génica , Genes , Vectores Genéticos , Interleucina-7/genética , Masculino , ARN Mensajero/genética , Ratas , Ratas Endogámicas Lew , RetroviridaeRESUMEN
The sponge allograft model of Roberts & Hayry and of Ascher et al. was modified by introducing bone marrow cells into uncoated sponges 7 days after the sponge was implanted into the mouse. The number and cellular composition of the response in the sponges was essentially the same whether the stimulus was allogeneic or syngeneic bone marrow. However, the allogeneically stimulated sponge derived cells demonstrated allospecific cell-mediated lympholysis at very low effector-to-target ratios. Similar cytotoxic activity was difficult to demonstrate in splenic cells from the same animal tested concurrently. The cytotoxic cells were shown to be Lyt-1-, 2+ and Thy 1+. For a bone marrow impregnated sponge the peak T cell killing was reached after 12 days whereas peak killing occurred on day 14 in peritoneal cell infiltrated sponges as utilized by Ascher et al. Our model was developed to study the response to various antigenic stimuli placed into the sponge. This variation of the sponge allograft model should permit the study of a local cellular immune response to a number of antigens.
Asunto(s)
Trasplante de Médula Ósea , Citotoxicidad Inmunológica , Modelos Biológicos , Trasplante Homólogo , Uretano , Animales , Anticuerpos Monoclonales , Inmunidad Celular , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Bazo/inmunología , Linfocitos T Citotóxicos/clasificación , Linfocitos T Citotóxicos/inmunologíaRESUMEN
If residual cancer cells in harvested bone marrow could be marked and subsequently detected in patients at relapse, valuable information would be obtained about the source of recurrent disease after autologous marrow transplantation. If normal progenitor cells were also marked, the study would provide useful data on the susceptibility of these human cells to gene transfer and their capacity to express newly introduced genes. We transferred the neomycin-resistance gene (NeoR) into bone marrow cells harvested from 20 children with acute myeloid leukemia (n = 12) or neuroblastoma (n = 8) in clinical and cytological remission using a retrovirus vector. The cells were then returned to the patients as part of an autologous bone marrow transplantation protocol. Two AML and three neuroblastoma patients have relapsed. In all, the resurgent cells contained the NeoR marker by analysis with PCR. These results prove that so-called remission marrow can contribute to relapse in patients who receive autologous transplants. The gene marking technique is now being used to evaluate techniques of pretransplant purging.
Asunto(s)
Trasplante de Médula Ósea/patología , Células Clonales , Técnicas de Transferencia de Gen , Células Madre Hematopoyéticas/citología , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/patología , Subgrupos Linfocitarios/patología , Neuroblastoma/diagnóstico , Neuroblastoma/patología , Recurrencia , Trasplante AutólogoRESUMEN
Retroviral vectors have provided a means for the introduction of functioning exogenous genes into the hematopoietic system of whole animals. Although these vectors are quite efficient in the mouse model, when applied to non-murine in vivo systems, the efficiency of gene transfer has diminished to impractical levels. Since in vivo analyses are expensive and time consuming, in vitro models have been developed to speed the evaluation of alternative protocols. Using in vitro colony assays, three approaches were evaluated for their ability to improve the infectivity of hematopoietic progenitor cells with retroviral vectors. Exogenously applied hematopoietic growth factors increased the proportion of hematopoietic colonies in vitro up to an average of 5 fold. When alternative sources of progenitors, such as fetal cord blood, were used, improvements in infection efficiency were also obtained. Finally, evidence was acquired suggesting that xenotropic packaging of vectors also improved infection efficiency.
Asunto(s)
Ingeniería Genética/métodos , Células Madre Hematopoyéticas/fisiología , Retroviridae/genética , Animales , Perros , Vectores Genéticos , Sustancias de Crecimiento/farmacología , Haplorrinos , Ratones , Receptores Virales/fisiología , Linfocitos T/fisiologíaAsunto(s)
Terapia Genética , Vectores Genéticos , Retroviridae/aislamiento & purificación , Replicación Viral , Células 3T3 , Animales , Genes Virales , Haplorrinos , Humanos , Leucemia Experimental/microbiología , Ganglios Linfáticos/microbiología , Linfoma/microbiología , Ratones , Virus de la Leucemia Murina de Moloney/genética , Retroviridae/genética , Retroviridae/patogenicidad , Retroviridae/fisiologíaAsunto(s)
Biomarcadores/análisis , Purgación de la Médula Ósea , Trasplante de Médula Ósea , Busulfano , Ciclofosfamida , Marcadores Genéticos , Interleucina-2 , Leucemia Mieloide/terapia , Mesna , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Enfermedad Aguda , Adolescente , Secuencia de Bases , Separación Celular , Niño , Preescolar , Protocolos Clínicos , Terapia Combinada , Resistencia a Medicamentos , Estudios de Evaluación como Asunto , Vectores Genéticos , Células Madre Hematopoyéticas , Humanos , Factores Inmunológicos , Lactante , Consentimiento Informado , Kanamicina Quinasa , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Proteínas Recombinantes de Fusión , Inducción de Remisión , Proyectos de Investigación , Retroviridae/genética , Seguridad , Trasplante AutólogoRESUMEN
Nanoparticles consisting of single molecules of DNA condensed with polyethylene glycol-substituted lysine 30-mers efficiently transfect lung epithelium following intrapulmonary administration. Nanoparticles formulated with lysine polymers having different counterions at the time of DNA mixing have distinct geometric shapes: trifluoroacetate or acetate counterions produce ellipsoids or rods, respectively. Based on intracytoplasmic microinjection studies, nanoparticle ellipsoids having a minimum diameter less than the 25 nm nuclear membrane pore efficiently transfect non-dividing cells. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids, whereas the 8-11 nm diameter of rod-like particles is smaller than the nuclear pore diameter. In mice, up to 50% of lung cells are transfected after dosing with a rod-like compacted 6.9 kbp lacZ expression plasmid, and correction of the CFTR chloride channel was observed in humans following intranasal administration of a rod-like compacted 8.3 kbp plasmid. To further investigate the potential size and shape limitations of DNA nanoparticles for in vivo lung delivery, reporter gene activity of ellipsoidal and rod-like compacted luciferase plasmids ranging in size between 5.3 and 20.2 kbp was investigated. Equivalent molar reporter gene activities were observed for each formulation, indicating that microinjection size limitations do not apply to the in vivo gene transfer setting.
Asunto(s)
Fibrosis Quística/terapia , Terapia Genética/métodos , Pulmón/enzimología , Plásmidos/genética , Transfección/métodos , Línea Celular , Fibrosis Quística/metabolismo , Células Epiteliales/enzimología , Expresión Génica , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Humanos , Luciferasas/análisis , Luciferasas/genética , Microscopía Electrónica de Transmisión , Nanoestructuras , NanotecnologíaRESUMEN
Retroviral mediated gene transfer has proven to be an effective method of transferring genetic material into many different mammalian cells. Presented in this short review is a history of retroviral-mediated gene transfer as it relates to hematopoietic- and blood-derived cells. Gene transfer into mouse, primate, and fetal sheep and primate hematopoietic stem cells is discussed. Gene transfer into lymphocytes, in particular tumor-infiltrating lymphocytes, is also addressed. The use of such lymphocytes in ongoing and submitted clinical protocols utilizing retroviral-mediated gene transfer are presented.
Asunto(s)
Adenosina Desaminasa/deficiencia , Terapia Genética/métodos , Adenosina Desaminasa/genética , Animales , Trasplante de Médula Ósea/métodos , Trasplante de Tejido Fetal/métodos , Humanos , Ratones , Primates , Retroviridae/genética , TransfecciónRESUMEN
During the embryonic development of chick calvaria (membranous cranial bones), the relative rate of procollagen synthesis increased from about 12% of total protein synthesis on Day 10 to about 65% on Day 17. This increase is due to a 1.7-fold increase in the absolute rate of procollagen synthesis and a 3-fold decrease in the synthesis of noncollagenous proteins. The increase in procollagen synthesis is directly proportional to an increase in procollagen mRNA content per cell as measured either by cell-free translation or by hybridization with complementary DNA. The results indicate that translational control of procollagen mRNA does not play a substantial role during calvaria development and that the specialization in the synthesis of this protein is largely due to the loss or inactivation of mRNAs for noncollagenous proteins.
Asunto(s)
Huesos/metabolismo , Procolágeno/biosíntesis , ARN Mensajero/metabolismo , Animales , Embrión de Pollo , Cinética , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/aislamiento & purificaciónRESUMEN
An inexpensive, easily performed enzyme-linked immunosorbent assay (ELISA) was developed to measure specific IgG, IgA, and IgM antibodies to the common antigens Escherichia coli, diphtheria-tetanus toxoid, and tetanus toxoid. Normal values were established. Classical antibody deficiency disease states were confirmed and delineated by these assays. Additionally, several instances were discovered when functional antibody levels were abnormal when the serum immunoglobulin levels were normal. The use of ELISA assays for antibodies to common antigens provides a useful technique to measure and monitor isotype responses of the humoral immune system.
Asunto(s)
Anticuerpos/análisis , Ensayo de Inmunoadsorción Enzimática , Adulto , Especificidad de Anticuerpos , Toxoide Diftérico/inmunología , Escherichia coli/inmunología , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Síndromes de Inmunodeficiencia/inmunología , Lactante , Masculino , Valores de Referencia , Toxoide Tetánico/inmunologíaRESUMEN
The role of the host cell cycle in determining the efficiency of infection with amphotropically packaged retroviral vectors was investigated in T lymphocytes and in fibroblasts. For T lymphocytes, the efficiency of infection with a retroviral vector was dependent on the cell cycle distribution of cells in culture at the time of exposure to the vector. When cultures enriched in the G0-G1 phase of the cell cycle (by serum starvation, aphidicolin treatment, or centrifugal elutriation) were exposed to retroviral vectors, the infection efficiency was severalfold lower than that in similar cultures enriched in the S, G2, and M phases. For fibroblasts, the efficiency of infection was not cell cycle dependent. These findings are relevant for studies with retrovirus-mediated gene transfer into hematopoietic tissues.
Asunto(s)
Vectores Genéticos , Interfase , Retroviridae/genética , Linfocitos T/microbiología , Animales , Fibroblastos/microbiología , Humanos , Ratones , Retroviridae/crecimiento & desarrollo , TransfecciónRESUMEN
In an effort to determine if viral envelope type influences the infectivity of human hematopoietic progenitor cells with retroviral vectors, we have pseudotyped the retroviral vector N2, which confers G418-resistance, in either an amphotropic or xenotropic envelope. Vector titres obtained by the pseudotype procedure were nearly two orders of magnitude lower than the titer obtained when N2 was packaged using the amphotropic PA317 packaging cell line. Despite its low titer, xenotropically pseudotyped N2 generated G418-resistant hematopoietic colonies at levels approaching those observed after bone marrow was infected using vector packaged using PA317 cells. These results suggest that manipulations of vector envelope may lead to improvements in the level of infection of human hematopoietic stem cells.
Asunto(s)
Médula Ósea/microbiología , Vectores Genéticos , Células Madre Hematopoyéticas/microbiología , Retroviridae/crecimiento & desarrollo , Animales , Células de la Médula Ósea , Línea Celular , Humanos , Ratones , Visón , Plásmidos , Retroviridae/genética , Retroviridae/ultraestructura , TransfecciónRESUMEN
A 4-month-old male received a T-lymphocyte-depleted haploidentical bone marrow transplant (BMT) for correction of severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency. Although previous haploidentical bone marrow transplants have been attempted in ADA-deficient SCID, complete reconstitution of both B-lymphocyte and T-lymphocyte function has not been obtained after a single transplant. In this patient, however, rapid, complete, and persistent engraftment occurred. Potential reasons for this successful reconstitution include the use of ablation by chemotherapy (busulfan, cyclophosphamide, and cytosine arabinoside), the in vitro technique of using monoclonal antibody (CT-2) and complement to deplete the donor cells of T lymphocytes, and the relative good health of the patient prior to the transplant. Further trials using this method of haploidentical BMT may prove it to be a successful method of immunologic reconstitution in ADA-deficient SCID patients for whom an HLA-identical marrow is not available.