RESUMEN
Definitive cure of an animal model of a human disease by gene transfer into hematopoietic stem cells has not yet been accomplished in the absence of spontaneous in vivo selection for transduced cells. Erythropoietic protoporphyria is a genetic disease in which ferrochelatase is defective. Protoporphyrin accumulates in erythrocytes, leaks into the plasma and results in severe skin photosensitivity. Using a mouse model of erythropoietic protoporphyria, we demonstrate here that ex vivo preselection of hematopoietic stem cells transduced with a polycistronic retrovirus expressing both human ferrochelatase and green fluorescent protein results in complete and long-term correction of skin photosensitivity in all transplanted mice.
Asunto(s)
Trasplante de Médula Ósea , Ferroquelatasa/genética , Terapia Genética , Trastornos por Fotosensibilidad/terapia , Porfiria Hepatoeritropoyética/terapia , Animales , Eritrocitos/metabolismo , Ferroquelatasa/biosíntesis , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Trastornos por Fotosensibilidad/sangre , Trastornos por Fotosensibilidad/patología , Porfiria Hepatoeritropoyética/sangre , Porfiria Hepatoeritropoyética/genética , Protoporfiria Eritropoyética , Protoporfirinas/sangre , Piel/patología , Trasplante IsogénicoRESUMEN
Because of a critical shortage in suitable organs, many patients with terminal liver disease die each year before liver transplantation can be performed. Transplantation of isolated hepatocytes has been proposed for the temporary metabolic support of patients awaiting liver transplantation or spontaneous reversion of their liver disease. A major limitation of this form of therapy is the present inability to isolate an adequate number of transplantable hepatocytes. A highly differentiated cell line, NKNT-3, was generated by retroviral transfer in normal primary adult human hepatocytes of an immortalizing gene that can be subsequently and completely excised by Cre/Lox site-specific recombination. When transplanted into the spleen of rats under transient immunosuppression, reversibly immortalized NKNT-3 cells provided life-saving metabolic support during acute liver failure induced by 90% hepatectomy.
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Técnicas de Cultivo de Célula/métodos , Trasplante de Células , Fallo Hepático Agudo/prevención & control , Hígado/citología , Proteínas Virales , Adulto , Animales , Antígenos Transformadores de Poliomavirus/genética , Diferenciación Celular , Línea Celular , Expresión Génica , Vectores Genéticos , Hepatectomía , Humanos , Integrasas/metabolismo , Hígado/metabolismo , Hígado/patología , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/patología , Fallo Hepático Agudo/terapia , Regeneración Hepática , Ratones , Ratones SCID , Ratas , Retroviridae/genética , Bazo/citología , TransfecciónRESUMEN
Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.
Asunto(s)
Anemia de Células Falciformes/terapia , Terapia Genética , Vectores Genéticos , Globinas/genética , VIH-1/genética , Anemia de Células Falciformes/genética , Animales , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Expresión Génica , Globinas/metabolismo , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Hemoglobina Falciforme/metabolismo , Humanos , Lentivirus/genética , Región de Control de Posición , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxihemoglobinas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Talasemia/genética , Talasemia/terapia , Transducción Genética , Transgenes , Globinas betaRESUMEN
Single-stranded oligonucleotides (SSOs) mediate gene repair of punctual chromosomal mutations at a low frequency. We hypothesized that enhancement of DNA binding affinity of SSOs by intercalating agents may increase the number of corrected cells. Several biochemical modifications of SSOs were tested for their capability to correct a chromosomally integrated and mutated GFP reporter gene in human 293 cells. SSOs of 25 nucleotide length conjugated with acridine at their 5' end increased the efficiency of gene correction up to 10-fold compared to nonmodified SSOs. Acridine and psoralen conjugates were both evaluated, and acridine-modified SSOs were the most effective. Conjugation with acridine at the 3' end of the SSO inhibited gene correction, whereas flanking the SSO by acridine on both sides provided an intermediate level of correction. These results suggest that increasing the stability of hybridization between SSO and its target without hampering a 3' extension improves gene targeting, in agreement with the "annealing-integration" model of DNA repair.
Asunto(s)
Acridinas/metabolismo , Sustancias Intercalantes/metabolismo , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Reparación del Gen Blanco/métodos , Ficusina/metabolismo , Terapia Genética , Humanos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , TransfecciónRESUMEN
Expression of experimental constructs in mammalian cells or transgenic animals is difficult to control because it is markedly influenced by position effects. This has limited both the analysis of cis -DNA regulatory elements for transcription and replication, and the physiological analysis of proteins expressed from transgenes. We report here two new methods based on the concept of recombinase-mediated cassette exchange (RMCE) to perform site-specific chromosomal integration. The first method permits the exchange of a negative selectable marker pre-localized on the chromosome with a transgene via a CRE-mediated double recombination between inverted Lox sites. Integration efficiency is close to 100 % of negatively selected mouse erythroleukemia cells and ranges from 10 to 50 % in embryonic stem cells. The second method allows RMCE with no selection at all except for cells that have taken up plasmid transiently. While less efficient, this technique permits novel experimental approaches. We find that integration of a transgene at a given genomic site leads to reproducible expression. RMCE should be useful to develop artificial genetic loci that impart specific and reproducible regulation of transgenes in higher eukaryotes. This should facilitate the analysis of cis -regulatory DNA elements governing expression and position effects, improve our control over the physiological effects of transgenes, and accelerate the development of animal models for complex human diseases.
Asunto(s)
Cromosomas/genética , Marcadores Genéticos/genética , Integrasas/metabolismo , Mutagénesis Insercional/métodos , Mutagénesis Sitio-Dirigida/genética , Proteínas Virales , Animales , Sitios de Ligazón Microbiológica/genética , Células Clonales/metabolismo , Clonación Molecular , Expresión Génica , Genes Reporteros/genética , Vectores Genéticos/genética , Genoma , Ratones , Mutagénesis Insercional/genética , Recombinación Genética/genética , Células Madre/metabolismo , Transfección , Transgenes/genética , Células Tumorales CultivadasRESUMEN
Retroviral transduction of human hematopoietic stem cells is still limited by lack of information about conditions that will maximize stem cell self-renewal divisions in vitro. To address this, we first compared the kinetics of entry into division of single human CD34+CD38- cord blood (CB) cells exposed in vitro to three different flt3-ligand (FL)-containing cytokine combinations. Of the three combinations tested, FL + hyperinterleukin 6 (HIL-6) yielded the least clones and these developed at a slow rate. With either FL + Steel factor (SF) + HIL-6 + thrombopoietin (TPO) or FL + SF + interleukin 3 (IL-3) + IL-6 + granulocyte-colony-stimulating factor (G-CSF), >90% of the cells that formed clones within 6 days undertook their first division within 4 days, although not until after 24 hours. These latter two, more stimulatory, cytokine combinations then were used to assess the effect of duration of cytokine exposure on the efficiency of transducing primitive CB cells with a gibbon ape leukemia virus-pseudotyped murine retroviral vector containing the enhanced green fluorescent protein (GFP) cDNA and the neomycin resistance gene. Fresh lin- CB cells exposed once to medium containing this virus plus cytokines on fibronectin-coated dishes yielded 23% GFP+ CD34+ cells and 52-57% G418-resistant CFC when assessed after 2 days. Prestimulation of the target cells (before exposing them to virus) with either the four or five cytokine combination increased their susceptibility. In both cases, the effect of prestimulation assessed using the same infection protocol was maximal with 2 days of prestimulation and resulted in 47-54% GFP+ CD34+ cells and 67-69% G418-resistant CFC. Repeated daily addition of new virus (up to three times), with assessment of the cells 2 days after the last addition of fresh virus, gave only a marginal improvement in the proportion of transduced CD34+ cells and CFC, but greatly increased the proportion of transduced LTC-IC (from 40% to >99%). Transplantation of lin- CB cells transduced using this latter 6-day protocol into NOD/SCID mice yielded readily detectable GFP+ cells in 10 of 11 mice that were engrafted with human cells. The proportion of the regenerated human cells that were GFP+ ranged from 0.2-72% in individual mice and included both human lymphoid and myeloid cells in all cases. High-level reconstitution with transduced human cells was confirmed by Southern blot analysis. These findings demonstrate that transplantable hematopoietic stem cells in human CB can be reproducibly transduced at high efficiency using a 6-day period of culture in a retrovirus-containing medium with either FL + SF + HIL-6 + TPO or FL + SF + IL-3 + IL-6 + G-CSF in which virus is added on the third, fourth, and fifth day.
Asunto(s)
Antígenos CD , Sangre Fetal/citología , Técnicas de Transferencia de Gen , Proteínas de la Membrana/genética , Retroviridae/genética , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Animales , Antígenos CD34/análisis , Antígenos de Diferenciación/análisis , División Celular , Sangre Fetal/inmunología , Vectores Genéticos , Humanos , Inmunofenotipificación , Recién Nacido , Glicoproteínas de Membrana , Ratones , Ratones Endogámicos NOD , Ratones SCID , NAD+ Nucleosidasa/análisis , Transducción GenéticaRESUMEN
The human muscle-specific phosphoglycerate mutase encoding gene (PGAM-M) has been cloned from a genomic cosmid library and sequenced. The sequence corresponding to the coding region was evaluated and revised by sequencing of the protein itself, fully confirming our results. The amino acid sequence of the M isozyme presented a 80.6% homology with the B isozyme (non-muscle-specific isozyme), a value higher than previously reported. The PGAM-M gene is composed of three exons, which consist of 454, 180 and 202 bp, respectively, and are separated by two introns of 103 bp and approx. 5.6 kb, respectively. Comparison of the structure of the human PGAM-M gene with that coding for human bisphosphoglycerate mutase, an erythroid-specific enzyme belonging to the same multifunctional enzyme family, revealed that the location of the second intron is similar in each gene and corresponds to a tertiary subdomain in the spatial structure of the protein. The transcription start point (tsp) in the PGAM-M gene was identified by both primer extension and S1 nuclease-protection experiments. A TATA-box-like element was observed 29 bp upstream from the tsp; the sequence ATTGG, inverse/complementary to CCAAT-box, was found 40 bp upstream from the supposed TATA box. No muscle-specific consensus sequences could be detected in the 5'-untranslated region. Only one polyadenylation AATAAA signal was observed in the short 3'-untranslated region (43 bp long). Finally, only one copy of this gene is present in the human genome instead of the several copies found for the PGAM-B gene, suggesting the possible evolutionary origin of the muscle subunit in a modified copy of the PGAM-B gene.
Asunto(s)
Bisfosfoglicerato Mutasa/genética , Isoenzimas/genética , Músculos/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Biológica , Bisfosfoglicerato Mutasa/metabolismo , Clonación Molecular , Secuencia de Consenso , Cósmidos , Eritrocitos/enzimología , Exones , Genes , Humanos , Intrones , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Poli A , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
We have constructed two fusion proteins, DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3, in which the receptor-binding domain of diphtheria toxin is replaced by mouse interleukin-3 (IL-3). Cytotoxic activity of the fusion toxins was observed on three out of six cell lines assayed. This toxicity was mediated through binding to the IL-3 receptor as it was inhibited in a dose-dependent manner with murine IL-3 or anti-IL-3 neutralizing antibodies. DAB389-(Gly4Ser)2-mIL-3 was up to 5 times more toxic than DAB389-mIL-3, depending on the cell line (0.8 x 10(-10) M < IC50 < 3 x 10(-10) M). These proteins can be used for the detection of IL-3 receptors on mouse cells and should allow for the selective elimination of IL-3 receptor-positive pluripotent hematopoietic stem cells prior to bone marrow transplantation.
Asunto(s)
Toxina Diftérica/química , Interleucina-2/química , Interleucina-3/química , Receptores de Interleucina-3/efectos de los fármacos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Toxina Diftérica/metabolismo , Toxina Diftérica/toxicidad , Interleucina-2/metabolismo , Interleucina-2/toxicidad , Interleucina-3/metabolismo , Interleucina-3/toxicidad , Ratones , Pliegue de Proteína , Receptores de Interleucina-3/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidadRESUMEN
PURPOSE: To determine whether human retinal pigment epithelium (RPE) can be modified by retroviral-mediated gene transfer and to monitor the human RPE cells in the subretinal space of living rabbits with scanning laser ophthalmoscopy (SLO). METHODS: Cultured human fetal retinal pigment epithelium (HFRPE) was exposed to green fluorescent protein (GFP)-transducing retroviral vectors, Moloney murine leukemia virus, and lentivirus. The cultured cells were followed by fluorescence microscopy. Suspensions of GFP-expressing HFRPE were transplanted into the subretinal space of pigmented rabbits, and the transplant sites were examined by SLO for fluorescence, including fluorescein and indocyanine green angiography. The rabbits were euthanatized at different times after transplantation, and the retinas were studied histologically. RESULTS: Retroviral gene transfer can introduce a foreign gene such as GFP into cultured HFRPE. Gene expression is maintained in cultured RPE for at least 3 months. The lentiviral vector transduced both nondividing and dividing cells; the Moloney vector only transduced the latter. GFP-expressing cells can be followed in the living retina. Their changes reflect the rejection response followed histologically. CONCLUSIONS: Cultured HFRPE could be transduced to express GFP for long periods of time by retroviral gene transfer. GFP allowed retinal transplants and gene expression to be monitored in vivo. These results provide a model for potential ex vivo gene therapy in the subretinal space.
Asunto(s)
Trasplante de Tejido Fetal , Técnicas de Transferencia de Gen , Proteínas Luminiscentes/metabolismo , Epitelio Pigmentado Ocular/trasplante , Retina/cirugía , Retroviridae/genética , Animales , Células Cultivadas , Virus Defectuosos , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Oftalmoscopía , Epitelio Pigmentado Ocular/metabolismo , Epitelio Pigmentado Ocular/virología , Conejos , Retina/citología , Retina/metabolismo , Retroviridae/metabolismoRESUMEN
We are focusing on the development of complex retroviral vectors containing human beta-globin gene and beta-LCR for the gene therapy of sickle cell disease and beta-thalassemias. First generation vectors containing mutated splice-sites to insure stability of proviral transfer enabled long-term reconstitution in 10/12 transplanted mice for a least 8 months with high expression levels in 2 out of 3 mice analyzed (5% and 20% murine beta). Transfer and expression were also achieved in secondary recipients (range: 3-11% murine beta). Position independent expression was not observed. In an effort to increase the efficiency of gene transfer and obtain complete reconstitution of recipient mice with exclusively transduced cells while enriching for proviral integration into active chromatin regions, we have incorporated a cassette expressing CD24 or the green fluorescent protein (GFP). Stable transfer to murine bone marrow cells allowed efficient FACS-sorting of pure populations of transduced cells. A family of vectors based on these principles and containing segments of gamma- or delta-globin genes were also designed for systematic analysis of their anti-sickling properties.
Asunto(s)
Anemia de Células Falciformes/terapia , Terapia Genética/métodos , Globinas/genética , Talasemia beta/terapia , Anemia de Células Falciformes/genética , Animales , Mapeo Cromosómico , Técnicas de Transferencia de Gen , Humanos , Región de Control de Posición , Ratones , Regiones Promotoras Genéticas , Retroviridae , Talasemia beta/genéticaRESUMEN
A worldwide shortage of donor livers is a limiting factor of the clinical application of hepatocyte transplantation (HTX). To resolve this issue, we focused on a reversible immortalization system that allows temporary expansion of primary hepatocyte populations by transfer of an oncogene that can be subsequently excised. As a preliminary test toward this goal, we examined the efficacy of Cre/loxP site-specific recombination in a transformed human liver cell line, HepG2. The present study utilized retroviral transfer of a prototypical immortalizing gene, simian virus 40 large T antigen (SV40Tag), flanked by a pair of loxP recombination targets and adenovirus-mediated Cre/loxP recombination. Here we report that complete elimination of the retroviral transferred oncogene was achieved by site-specific recombination using a replication-deficient recombinant adenovirus vector producing Cre recombinase (Ad-Cre).
Asunto(s)
Hepatocitos/virología , Integrasas/metabolismo , Recombinación Genética , Proteínas Virales , Adenoviridae/genética , Animales , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/inmunología , Antígenos Transformadores de Poliomavirus/metabolismo , Western Blotting , División Celular , Línea Celular Transformada , Virus Defectuosos/genética , Técnica del Anticuerpo Fluorescente , Ganciclovir/farmacología , Expresión Génica , Gentamicinas/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Integrasas/genética , Ratones , Ratones SCID , Neoplasias Experimentales/patología , ARN Mensajero/biosíntesis , Retroviridae/genética , Transducción GenéticaRESUMEN
Current clinical reports have indicated that hepatocyte transplantation (HTX) could be used in patients with liver failure and in children with liver-based metabolic diseases. One of the major limiting factors of HTX is a serious shortage of donor livers for hepatocyte isolation. To address this issue, we immortalized adult human hepatocytes with a retroviral vector SSR#69 expressing the genes of simian virus 40 large T antigen and herpes simplex virus-thymidine kinase simultaneously. One of the resulting clones, NKNT-3, grew steadily in chemically defined serum-free medium without any obvious crisis and showed the gene expression of differentiated liver functions. Under the administration of 5 microM ganciclovir, NKNT-3 cells stopped proliferation and died in in vitro experiments. We have established a tightly regulated immortal human hepatocyte cell line. The cells could allow the need for immediate availability of consistent and functionally uniform cells in sufficient quantity and adequate quality.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Línea Celular , Técnicas de Transferencia de Gen , Hepatocitos/fisiología , Simplexvirus/enzimología , Timidina Quinasa/genética , Antivirales/farmacología , Trasplante de Células/métodos , Medio de Cultivo Libre de Suero , Ganciclovir/farmacología , Expresión Génica , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/trasplante , Humanos , Inmunohistoquímica , Hígado/fisiología , Trasplante de Hígado , Hígado Artificial , Retroviridae/genética , Retroviridae/metabolismoRESUMEN
An ideal alternative to the primary human hepatocytes for hepatocyte transplantation would be to use a clonal cell line that grows economically in culture and exhibits the characteristics of differentiated, nontransformed hepatocytes following transplantation. The purpose of the present studies was to establish a reversibly immortalized human hepatocyte cell line. Human hepatocytes were immortalized with a retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40Tag) gene flanked by a pair of loxP recombination targets. One of the resulting clones, NKNT-3, showed morphological characteristics of liver parenchymal cells and expressed the genes of differentiated liver functions. NKNT-3 cells offered unlimited availability. After an adenoviral delivery of Cre recombinase and subsequent differential selection, efficient removal of SV40Tag from NKNT-3 cells was performed. Here we represent that elimination of the retrovirally transferred SV40Tag gene can be excised by adenovirus-mediated site-specific recombination.
Asunto(s)
Línea Celular , Técnicas de Transferencia de Gen , Hepatocitos/fisiología , Integrasas/metabolismo , Proteínas Virales/metabolismo , Trasplante de Células , Medio de Cultivo Libre de Suero , Vectores Genéticos , Hepatocitos/trasplante , Humanos , Hígado/fisiología , Recombinación Genética , Retroviridae/genética , Retroviridae/metabolismo , Transducción GenéticaRESUMEN
Hepatocyte transplantation (HTX) could be an attractive treatment for patients with liver failure and liver-based metabolic disease. Human primary hepatocytes are ideal in this modality, but the shortage of human livers available for hepatocyte isolation severely limits the use of this form of therapy. A tightly regulated human hepatocyte cell line that grows economically in culture and exhibits differentiated liver functions would be an attractive alternative to the primary human hepatocytes. To test the feasibility, human hepatocytes were immortalized by a retroviral vector expressing simian virus 40 large T antigen and herpes simplex virus-thymidine kinase. A highly differentiated immortal hepatocyte line NKNT-3 was established. NKNT-3 cells grew in chemically defined serum-free medium, retained highly differentiated liver functions, and were sensitivity to ganciclovir as a prodrug. Essentially unlimited availability of NKNT-3 cells may be clinically useful for HTX and bioartificial liver.
Asunto(s)
Trasplante de Hígado , Hígado/citología , Trasplantes , Antígenos Virales , Línea Celular , Supervivencia Celular , Estudios de Factibilidad , Humanos , Hepatopatías/terapia , Virus 40 de los Simios/inmunología , Timidina QuinasaRESUMEN
Currently patients with liver failure have been treated with a various liver support systems including a whole liver perfusion, a non-biological artificial liver, and a biohybrid artificial liver. In a hepatocyte-based bioreactor, porcine hepatocytes or transformed human liver tumor cells have been utilized because of the ease of preparation. According to the clinical data reported as of now, satisfactory results have not been obtained from the use of currently available liver support devices. One of the problems is limited availability of primary human liver cells for developing live support systems because of the shortage of human liver. To resolve this issue, human hepatocytes were immortalized with a retroviral vector SSR#69 which contained the genes of simian virus 40 large T antigen (SV40Tag) and herpes simplex virus-thymidine kinase (HSV-TK). One of the immortal cell lines, NKNT-3, showed the gene expression of differentiated liver functions, grew steadily in chemically defined serum-free CS-C medium, and doubled in number in about 48 hours. Essentially unlimited availability of NKNT-3 cells supports their clinical use for liver support devices. To realize the high density culture of NKNT-3 cells in a bioartificial liver device, we have developed cellulose microspheres (CMS) which contain cell adhesive GRGDS (Gly-Arg-Gly-Asp-Ser) peptides. Within 24 hours after starting a stirring suspension culture, GRGDS-CMS efficiently immobilized NKNT-3 cells. An electron microscopic examination demonstrated that NKNT-3 cells attached on GRGDS-CMS had well-developed mitochondria, rough reticulums, and villous extensions. In this article, we review the history of extracorporeal liver support systems and describe an attractive strategy for developing a novel extracorporeal liver assist device using NKNT-3 cells and GRGDS-coated cellulose microspheres.
Asunto(s)
Hepatocitos/citología , Hígado Artificial , Animales , Antígenos Transformadores de Poliomavirus , División Celular , Línea Celular , Celulosa , Humanos , Microesferas , Oligopéptidos , PorcinosRESUMEN
Transient support using a hybrid artificial liver (HAL) device is a promising treatment for the patients with acute liver failure. Primary human hepatocytes are an ideal source for HAL therapy; however, the number of human livers available for hepatocyte isolation is limited by competition for use in whole organ transplantation. To overcome this problem, we previously established a highly differentiated human fetal hepatocyte cell line OUMS-29. Considering the potential risk when using these genetically engineered cells in humans, additional safeguards should be added to make the cells more clinically useful. In this work, the herpes simplex virus thymidine kinase (HSVtk) gene was retrovirally introduced into OUMS-29 cells. One of the HSVtk-expressed clones, OUMS-29/thymidine kinase (TK), grew in chemically defined serum free medium and expressed the genes of albumin, asialoglycoprotein receptor, glutamine synthetase, glutathione-S-transferase pi, and blood coagulation factor X. In vitro sensitivity of the cells to ganciclovir was evaluated. Intrasplenic transplantation of 50 x 10(6) OUMS-29/TK cells prolonged the survival of 90% hepatectomized rats compared with medium injection alone (control). In the present study, we have established highly differentiated immortalized human hepatocytes with tight regulation. The cells may be clinically useful for HAL treatment.
Asunto(s)
Hepatocitos/citología , Hepatocitos/enzimología , Simplexvirus/enzimología , Simplexvirus/genética , Timidina Quinasa/genética , Albúminas/biosíntesis , Animales , Diferenciación Celular , Línea Celular , Resistencia a Medicamentos/genética , Ganciclovir/farmacología , Expresión Génica , Genes Virales , Hepatocitos/trasplante , Hepatocitos/virología , Humanos , Fallo Hepático Agudo/terapia , Hígado Artificial , Masculino , Ratones , Ratones Desnudos , Ratas , Ratas Endogámicas Lew , Transducción GenéticaRESUMEN
A hybrid artificial liver (HAL) could be used to treat acute liver failure or to serve as a temporary support until orthotopic liver transplantation is available. Primary human hepatocytes are ideal as a source of hepatic function in a HAL device. However, the worldwide shortage of human livers available for hepatocyte isolation severely limits this form of therapy. A possible alternative is to use a tightly regulated cell line that can be economically grown in culture to have differentiated liver function. In this work, human hepatocytes were immortalized with a retroviral vector SSR#69 expressing the genes of simian virus 40 large T antigen and herpes simplex virus-thymidine kinase. One of the resulting clones, NKNT-3 , showed the gene expression of differentiated liver function and were sensitive to the antiviral agent ganciclovir. When transplanted into the spleen of rats subjected to 90% hepatectomy, NKNT-3 cells prolonged the survival of 90% hepatectomized rats. The cells provide the advantages of unlimited availability, sterility, uniformity, and freedom from pathogens. This work represents a potential novel strategy for resolving the organ shortage that currently limits the use of primary human hepatocytes to develop a HAL.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Hepatocitos/citología , Hepatocitos/inmunología , Lesión Renal Aguda/terapia , Animales , Diferenciación Celular , División Celular , Línea Celular , Celulosa , Resistencia a Medicamentos/genética , Ganciclovir/farmacología , Expresión Génica , Técnicas de Transferencia de Gen , Genes Virales , Vectores Genéticos , Hepatocitos/trasplante , Hepatocitos/virología , Humanos , Hígado Artificial , Ratones , Ratones SCID , Microesferas , Retroviridae/genética , Trasplante HeterólogoAsunto(s)
Antígenos Transformadores de Poliomavirus/genética , Hepatocitos/citología , Virus 40 de los Simios/genética , Adulto , Antígenos Transformadores de Poliomavirus/análisis , Receptor de Asialoglicoproteína , Línea Celular Transformada , Transformación Celular Viral , Factor X/genética , Técnica del Anticuerpo Fluorescente Indirecta , Vectores Genéticos , Glucuronosiltransferasa/genética , Glutamato-Amoníaco Ligasa/genética , Glutatión Transferasa/genética , Humanos , Reacción en Cadena de la Polimerasa , Receptores de Superficie Celular/genética , Retroviridae , Albúmina Sérica/genética , Secuencias Repetidas TerminalesRESUMEN
BACKGROUND: Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids. METHODS: Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target. RESULTS: Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease. CONCLUSIONS: Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.
Asunto(s)
ADN Circular/genética , Desoxirribonucleasas de Localización Especificada Tipo II/farmacología , Vectores Genéticos/análisis , Proteínas Fluorescentes Verdes/genética , Recombinación Genética , Animales , Células CHO , Cricetinae , Marcación de Gen/métodos , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas de Saccharomyces cerevisiae , TransfecciónRESUMEN
A procedure of reversible immortalization of primary cells was devised by retrovirus-mediated transfer of an oncogene that could be subsequently excised by site-specific recombination. This study focused on the early stages of immortalization: global induction of proliferation and life span extension of cell populations. Comparative analysis of Cre/LoxP and FLP/FRT recombination in this system indicated that only Cre/LoxP operates efficiently in primary cells. Pure populations of cells in which the oncogene is permanently excised were obtained, following differential selection of the cells. Cells reverted to their preimmortalized state, as indicated by changes in growth characteristics and p53 levels, and their fate conformed to the telomere hypothesis of replicative cell senescence. By permitting temporary and controlled expansion of primary cell populations without retaining the transferred oncogene, this strategy may facilitate gene therapy manipulations of cells unresponsive to exogenous growth factors and make practical gene targeting by homologous recombination in somatic cells. The combination of retroviral transfer and site-specific recombination should also extend gene expression studies to situations previously inaccessible to experimentation.