RESUMO
Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.
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The subretinal injection protocol for the only approved retinal gene therapy (voretigene neparvovec-rzyl) includes air tamponade at the end of the procedure, but its effects on the subretinal bleb have not been described. In the present study, we evaluated the distribution of enhanced green fluorescent protein (EGFP) after subretinal injection of AAV2 in non-human primates (NHP) without (group A = 3 eyes) or with (group B = 3 eyes) air tamponade. The retinal expression of EGFP was assessed 1 month after subretinal injection with in vivo fundus photographs and fundus autofluorescence. In group A (without air), EGFP expression was limited to the area of the initial subretinal bleb. In group B (with air), EGFP was expressed in a much wider area. These data show that the buoyant force of air on the retina causes a wide subretinal diffusion of vector, away from the injection site. In the present paper, we discuss the beneficial and deleterious clinical effects of this finding. Whereas subretinal injection is likely to become more common with the coming of new gene therapies, the effects of air tamponade should be explored further to improve efficacy, reproducibility, and safety of the protocol.
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Adeno-associated virus (AAV) vectors are considered efficient vectors for gene transfer, as illustrated by recent successful clinical trials targeting retinal or neurodegenerative disorders. However, limitations as host immune responses to AAV capsid or transduction of limited regions must still be overcome. Here, we focused on locoregional (LR) intravenous perfusion vector delivery that allows transduction of large muscular areas and is considered to be less immunogenic than intramuscular (IM) injection. To confirm this hypothesis, we injected 6 cynomolgus monkeys with an AAV serotype 8 (AAV8) vector encoding for the highly immunogenic GFP driven by either a muscle-specific promoter (n = 3) or a cytomegalovirus (CMV) promoter (n = 3). We report that LR delivery allows long-term GFP expression in the perfused limb (up to 1 year) despite the initiation of a peripheral transgene-specific immune response. The analysis of the immune status of the perfused limb shows that LR delivery induces persisting inflammation. However, this inflammation is not sufficient to result in transgene clearance and is balanced by resident regulatory T cells. Overall, our results suggest that LR delivery promotes persisting transgene expression by induction of Treg cells in situ and might be a safe alternative to IM route to target large muscle territories for the expression of secreted therapeutic factors.
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Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents. Here, we address the role of the vitreous body as a potential barrier to AAV2/2 diffusion and transduction in the RGCs of dogs and macaques, two of the most relevant preclinical models. We intravitreally administered the AAV2/2 vector carrying the CMV-eGFP reporter cassette in dog and macaque eyes, either directly into the vitreous chamber or after complete vitrectomy, a surgical procedure that removes the vitreous body. Our findings suggest that the vitreous body appears to trap the injected vector, thus impairing the diffusion and transduction of AAV2/2 to inner retinal neurons. We show that vitrectomy before intravitreal vector injection is an effective means of overcoming this physical barrier, improving the transduction of RGCs in dog and macaque retinas. These findings support the use of vitrectomy in clinical trials of intravitreal gene transfer techniques targeting inner retinal neurons.
Assuntos
Terapia Genética , Vetores Genéticos/uso terapêutico , Células Ganglionares da Retina , Animais , Dependovirus/genética , Cães , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde , Humanos , Injeções Intravítreas , Macaca , Retina/patologia , Retina/transplante , Transdução Genética , VitrectomiaRESUMO
We previously reported that subretinal injection of AAV2/5 RK.cpde6ß allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Terapia Genética/métodos , Degeneração Retiniana/terapia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/deficiência , Dependovirus/genética , Modelos Animais de Doenças , Cães , Vetores Genéticos/administração & dosagem , Humanos , Retina/fisiopatologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologiaRESUMO
In a previous study, we identified TRIB1, a serine-threonine kinase-like molecule, as a biomarker of chronic antibody-mediated rejection of human kidneys when measured in peripheral blood mononuclear cells. Here, we focused our analysis on a specific subset of peripheral blood mononuclear cells that play a dominant role in regulating immune responses in health and disease, so-called CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). We isolated both human and murine Treg and non-Treg counterparts and analyzed TRIB1 and Foxp3 mRNA expression by quantitative PCR on the freshly isolated cells or following 24 h of activation. Physical interaction between the human TRIB1 and Foxp3 proteins was analyzed in live cell lines by protein complementation assay using both flow cytometry and microscopy and confirmed in primary freshly isolated human CD4(+)CD25(hi)CD127(-) Tregs by co-immunoprecipitation. Both TRIB1 and Foxp3 were expressed at significantly higher levels in Tregs than in their CD4(+)CD25(-) counterparts (p < 0.001). Moreover, TRIB1 and Foxp3 mRNA levels correlated tightly in Tregs (Spearman r = 1.0; p < 0.001, n = 7), but not in CD4(+)CD25(-) T cells. The protein complementation assay revealed a direct physical interaction between TRIB1 and Foxp3 in live cells. This interaction was impaired upon deletion of the TRIB1 N-terminal but not the C-terminal domain, suggesting an interaction in the nucleus. This direct interaction within the nucleus was confirmed in primary human Tregs by co-immunoprecipitation. These data show a direct relationship between TRIB1 and Foxp3 in terms of their expression and physical interaction and highlight Tribbles-1 as a novel binding partner of Foxp3 in Tregs.
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Fatores de Transcrição Forkhead/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Linfócitos T Reguladores/citologia , Animais , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/imunologia , Adesão Celular , Ciclo Celular , Proliferação de Células , Sobrevivência Celular , DNA Complementar/metabolismo , Teste de Complementação Genética , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucócitos Mononucleares/citologia , Camundongos , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Linfócitos T/citologia , Linfócitos T Reguladores/metabolismoRESUMO
Lentiviral vectors are efficient gene delivery vehicles for therapeutic and research applications. In contrast to oncoretroviral vectors, they are able to infect most nonproliferating cells. In the liver, induction of cell proliferation dramatically improved hepatocyte transduction using all types of retroviral vectors. However, the precise relationship between hepatocyte division and transduction efficiency has not been determined yet. Here we compared gene transfer efficiency in the liver after in vivo injection of recombinant lentiviral or Moloney murine leukemia viral (MoMuLV) vectors in hepatectomized rats treated or not with retrorsine, an alkaloid that blocks hepatocyte division and induces megalocytosis. Partial hepatectomy alone resulted in a similar increase in hepatocyte transduction using either vector. In retrorsine-treated and partially hepatectomized rats, transduction with MoMuLV vectors dropped dramatically. In contrast, we observed that retrorsine treatment combined with partial hepatectomy increased lentiviral transduction to higher levels than hepatectomy alone. Analysis of nuclear ploidy in single cells showed that a high level of transduction was associated with polyploidization. In conclusion, endoreplication could be exploited to improve the efficiency of liver-directed lentiviral gene therapy.
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Vetores Genéticos/metabolismo , Lentivirus/metabolismo , Fígado/virologia , Mitose , Poliploidia , Recombinação Genética , Animais , Núcleo Celular/virologia , Proliferação de Células , Endorreduplicação , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatectomia , Hepatócitos/efeitos dos fármacos , Imuno-Histoquímica , Lentivirus/genética , Fígado/efeitos dos fármacos , Masculino , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/metabolismo , Alcaloides de Pirrolizidina/farmacologia , Ratos , Ratos Endogâmicos F344 , Transdução GenéticaRESUMO
Precise control of transgene expression in a tissue-specific and temporally regulated manner is desirable for many basic and applied investigations gene therapy applications. This is important to regulate dose of transgene products and minimize unwanted effects. Previously described methods have employed tissue specific promoters, miRNA-based transgene silencing or tetR-KRAB-mediated suppression of transgene promoters. To improve on versatility of transgene expression control, we have developed expression systems that use combinations of a tetR-KRAB artificial transgene-repressor, endogenous miRNA silencing machinery and tissue specific promoters. Precise control of transgene expression was demonstrated in liver-, macrophage- and muscle-derived cells. Efficiency was also demonstrated in vivo in murine muscle. This multicomponent and modular regulatory system provides a robust and easily adaptable method for achieving regulated transgene expression in different tissue types. The improved precision of regulation will be useful for many gene therapy applications requiring specific spatiotemporal transgene regulation.
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Vetores Genéticos/genética , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/genética , Proteínas Repressoras/genética , Transcrição Gênica , Transgenes , Animais , Linhagem Celular , Linhagem Celular Tumoral , Inativação Gênica , Células HEK293 , Humanos , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células Musculares/metabolismo , Regiões Promotoras GenéticasRESUMO
Lentiviral vectors are promising tools for liver disease gene therapy, because they can achieve protracted expression of transgenes in hepatocytes. However, the question as to whether cell division is required for optimal hepatocyte transduction has still not been completely answered. Liver gene-transfer efficiency after in vivo administration of recombinant lentiviral vectors carrying a green fluorescent protein reporter gene under the control of a liver-specific promoter in mice that were either hepatectomized or treated with cholic acid or phenobarbital was compared. Phenobarbital is known as a weak inducer of hepatocyte proliferation, whereas cholic acid has no direct effect on the cell cycle. This study shows that cholic acid is able to prime hepatocytes without mitosis induction. Both phenobarbital and cholic acid significantly increased hepatocyte transduction six- to ninefold, although cholic acid did not modify the mitotic index or cell-cycle entry. However, the effect of either compound was weaker than that observed after partial hepatectomy. In no cases was there a correlation between the expression of cell-cycle marker and transduction efficiency. We conclude that priming of hepatocytes should be considered a clinically applicable strategy to enhance in vivo liver gene therapy with lentiviral vectors.
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Ácido Cólico/farmacologia , Terapia Genética/métodos , Vetores Genéticos/genética , Hepatócitos/efeitos dos fármacos , Fígado/metabolismo , Transdução Genética/métodos , Animais , Proliferação de Células/efeitos dos fármacos , Primers do DNA/genética , Proteínas de Fluorescência Verde/genética , Hematócrito , Imuno-Histoquímica , Lentivirus/genética , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenobarbital/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não ParamétricasRESUMO
Retroviral vectors have been used for several decades for the transfer of therapeutic genes to various cells or organs including the liver. Initial studies aimed at treating inherited liver deficiencies were carried out with murine oncoretroviral vectors either delivered directly to the organ or using an ex vivo strategy that entailed harvest of the hepatocytes, transduction during a culture phase and further reinfusion to the patient. However, although a clinical trial was performed in the early 1990s, a complete cure of animal models of metabolic diseases was rarely achieved. The advent of lentiviral vectors derived from HIV1 profoundly changed the field and this vector type now appears to be of the most attractive for liver directed gene therapy. Indeed, lentiviral vectors do not require complete cell division to transduce the target cells. There are however still bottlenecks that limit the clinical development of gene therapy using retroviral vectors. In the present review we will specifically focus on specific aspects such as the risk of insertional mutagenesis, the potential requirement of cell cycle activation to enhance transduction and the major issue of an immune response directed against the transgene as well as some specific aspects of ex vivo gene transfer. Finally we will briefly consider the future developments of these vectors made possible by the availability of new techniques in cell and molecular biology.
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Terapia Genética/métodos , Doenças Metabólicas/terapia , Retroviridae/genética , Animais , Ciclo Celular , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Lentivirus/genética , Fígado/metabolismo , Biologia Molecular/métodos , TransgenesRESUMO
BACKGROUND & AIMS: Crigler-Najjar type 1 (CN-I) is an inherited liver disease caused by an absence of bilirubin-uridine 5'-diphosphate-glucuronosyltransferase (UGT1A1) activity. It results in life-threatening levels of unconjugated bilirubin, and therapeutic options are limited. We used adult Gunn rats (an animal model of the disease) to evaluate the efficiency of lentiviral-based gene therapy to express UGT1A1 in liver. METHODS: Gunn rats were given intraportal injections of VSVG-pseudotyped lentiviral vectors that encode UGT1A1 under the control of a liver-specific transthyretin promoter (mTTR.hUGT1A1); this vector does not contain target sequences for miR-142, a microRNA that is expressed specifically in hematopoietic cells. Rats were also injected with the vector mTTR.hUGT1A1.142T, which contains 4 copies of the miR-142 target sequences; its messenger RNA should be degraded in antigen-presenting cells. Bilirubinemia was monitored, and the presence of transduced hepatocytes was analyzed by quantitative polymerase chain reaction. Vector expression was tested in vitro in rat hematopoietic cells. RESULTS: In Gunn rats, bilirubin levels normalized 2 weeks after administration of mTTR.hUGT1A1. However, hyperbilirubinemia resumed 8 weeks after vector administration, concomitant with the induction of an immune response. In contrast, in rats injected with mTTR-UGT1A1.142T, bilirubin levels normalized for up to 6 months and transduced cells were not eliminated. CONCLUSIONS: Lentiviral vectors that express UGT1A1 reduce hyperbilirubinemia in immunocompetent Gunn rats for at least 6 months. The immune response against virally expressed UGT1A1 can be circumvented by inclusion of miR-142 target sequences, which reduce vector expression in antigen-presenting cells. This lentiviral-based gene therapy approach might be developed to treat patients with CN-I.
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Síndrome de Crigler-Najjar/terapia , Terapia Genética/métodos , Vetores Genéticos , Glucuronosiltransferase/genética , Lentivirus/genética , Fígado/enzimologia , MicroRNAs/metabolismo , Animais , Anticorpos/sangue , Células Apresentadoras de Antígenos/imunologia , Bilirrubina/sangue , Biomarcadores/sangue , Síndrome de Crigler-Najjar/enzimologia , Síndrome de Crigler-Najjar/genética , Síndrome de Crigler-Najjar/imunologia , Modelos Animais de Doenças , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/imunologia , Células HeLa , Humanos , Masculino , Pré-Albumina/genética , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ratos , Ratos Gunn , Fatores de Tempo , Transdução GenéticaRESUMO
Lentiviral vectors can stably transduce hepatocytes and are promising tools for gene therapy of hepatic diseases. Although hepatocytes are accessible to blood-borne viral vectors through fenestrations of the hepatic endothelium, improved liver transduction after delivery of vectors to the blood stream is needed. As the normal endothelial fenestration and lentiviral vectors are similar in size (150 nm), we hypothesized that a transient increase in hepatic blood pressure may enhance in vivo gene transfer to hepatocytes. We designed a simple surgical procedure, by which the liver is temporarily excluded from blood flow. Lentiviral vectors were injected in a large volume to increase intrahepatic pressure. We demonstrated that in the Gunn rat, a model of Crigler-Najjar disease, the administration of low vector doses (corresponding to a multiplicity of infection of 0.2) by this procedure resulted in therapeutic correction of hyperbilirubinemia, without toxicity. The correction was sustained for 10 months (end of study). The same vector amounts yielded only partial correction after intraportal delivery. We believe that this new and clinically applicable strategy may broaden the range of genetic liver diseases accessible to gene therapy.
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Síndrome de Crigler-Najjar/terapia , Terapia Genética/métodos , Vetores Genéticos , Hiperbilirrubinemia/terapia , Lentivirus/genética , Fígado , Transdução Genética , Animais , Bilirrubina/sangue , Biomarcadores/sangue , Pressão Sanguínea , Síndrome de Crigler-Najjar/genética , Modelos Animais de Doenças , Hepatócitos , Fígado/irrigação sanguínea , Fígado/cirurgia , Testes de Função Hepática , Reação em Cadeia da Polimerase , Pressão , Ratos , Ratos GunnRESUMO
BACKGROUNDS AND AIMS: When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment. METHODOLOGY/PRINCIPAL FINDINGS: We used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats. CONCLUSIONS: Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.
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2-Acetilaminofluoreno/farmacologia , Fígado/lesões , Animais , Animais Recém-Nascidos , Antineoplásicos Fitogênicos/farmacologia , Linhagem da Célula , Proliferação de Células , Células Epiteliais/citologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Alcaloides de Pirrolizidina/farmacologia , Ratos , Ratos Endogâmicos F344 , Células-Tronco/citologia , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: In vivo adeno-associated virus (AAV) delivery to adult liver results in sustained expression of the transgene. However, it has been suggested that AAV delivery to the newborn liver may result in transient expression. In the present study, we analysed transgene expression after AAV8 delivery of a therapeutic or a marker gene to newborn rat liver. METHODS: Recombinant AAV 8 vectors carrying either the human UGT1A1 cDNA or the lacZ gene were injected intravenously in 2-day-old Gunn or Wistar rats. Serum bilirubin level was recorded in Gunn rats and beta-galactosidase expression was monitored by immunohistochemistry or enzyme activity. The molecular forms of AAV genome were analysed by the polymerase chain reaction and Southern blotting in whole liver and by the quantitative polymerase chain reaction in macroscopically dissected beta-galactosidase clusters. RESULTS: In Gunn rat, complete serum bilirubin normalization occurred after AAV delivery but hyperbilirubinemia resumed thereafter. Similarly, beta-galactosidase expression was maximum at day 7, but only a few (less than 1%) beta-galactosidase positive cells were recorded at 1 or 3 months. These cells gathered in small clusters and the AAV copy number was 75-fold higher in positive cell clusters than in the surrounding parenchyma. CONCLUSIONS: The results obtained in the present study show that in vivo AAV delivery to newborn rats results in transient expression in most hepatocytes. Expression of the trangene was persistent in small clusters of cells and preliminary data support the hypothesis that integration of the viral genome occurs in these clusters. Altogether, our data confirm the low efficiency of AAV vectors for gene therapy of liver diseases when delivered in the newborn period.
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DNA Recombinante/metabolismo , Dependovirus/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Fígado/metabolismo , Animais , Animais Recém-Nascidos , Bilirrubina/sangue , Southern Blotting , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/administração & dosagem , Glucuronosiltransferase/metabolismo , Humanos , Injeções , Reação em Cadeia da Polimerase , Ratos , Ratos Gunn , Fatores de Tempo , Integração Viral , beta-Galactosidase/metabolismoRESUMO
PURPOSE: Metabolic inherited liver diseases are attractive targets for gene therapy. Recombinant lentiviruses are very powerful viral vectors able to infect nonmitotic cells. We wanted to develop a new surgical approach to improve gene transfer in adult liver using low viral doses. MATERIALS AND METHODS: Adult rats were injected with 2.108 infectious particles of lentiviral vectors encoding the green fluorescent protein marker gene under control of a liver-specific promoter transthyretin. In the control group (n = 5), gene delivery was performed by inflow intraportal injection. In the surgical group (n = 5), liver was completely excluded from systemic circulation before viral injection in infrahepatic vena cava with high pressure. RESULTS: At day 9, transduction efficiency was 14.35% in the surgical group 3 and 0.39% in the control group (P = .016). At month 2, the number of transduced hepatocytes decreased in the most part of rats, except in half of rats in the surgical group. Antibodies against green fluorescent protein were detected in all rats at month 2, except one in the surgical group. CONCLUSIONS: We developed a new surgical approach allowing an efficient transduction of hepatocytes in adult rats using lentivirus at low viral doses. We have now to control the immune response to permit long-term expression of transgene.
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Técnicas de Transferência de Genes , Vetores Genéticos , Lentivirus/genética , Animais , Contagem de Células , Células Cultivadas , Feminino , Proteínas de Fluorescência Verde/genética , Hepatócitos/metabolismo , Imuno-Histoquímica , Fígado/irrigação sanguínea , Circulação Hepática , Masculino , Plasmídeos , Ratos , Ratos Gunn , Transdução Genética , Veias CavasRESUMO
alpha-fetoprotein (AFP) is a fetal protein specifically reexpressed in 50% of hepatocellular carcinomas. This protein could serve as a tumor-associated antigen for immunotherapy purpose. The aim of our work was to analyze the presence of AFP-specific T cell populations in peripheral blood mononuclear cells (PBMCs) from cirrhotic patients with or without hepatocellular carcinoma. Using peptide-major histocompatibility complex class I multimers, AFP-specific populations corresponding to 3 previously described human leukocyte antigen (HLA)-A*0201 major histocompatibility complex class I epitopes (AFP137, AFP158, and AFP325) were sorted magnetically from CD8 positive cells without prior stimulation with the target antigen. T cell populations specific for 1 peptide (AFP158) were frequent, whereas populations corresponding to peptide AFP137 were rare and absent for peptide AFP325. We also isolated and fully characterized T cell clones specific for AFP137 and AFP158 peptides. We show that these clones can be used to monitor dendritic cell loading with peptides and could be useful for future immunotherapy protocols.
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Antígenos HLA-A/imunologia , Separação Imunomagnética , Subpopulações de Linfócitos T/imunologia , alfa-Fetoproteínas/análise , alfa-Fetoproteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Testes Imunológicos de Citotoxicidade , Feminino , Antígenos HLA-A/isolamento & purificação , Antígenos HLA-A/metabolismo , Antígeno HLA-A2 , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/patologia , alfa-Fetoproteínas/imunologiaRESUMO
BACKGROUND/AIMS: To optimise vaccination strategies for immunotherapy in the liver, we have generated a line of transgenic mice expressing beta-Galactosidase downstream of the alpha-fetoprotein promoter (AFP/betaGal). METHODS: betaGal expression was documented by qRT-PCR, enzyme activity and immunohistochemistry. betaGal-specific CD8+ T-cell activation in mice immunised with various vectors was measured by interferon-gamma ELISpot. RESULTS: Like AFP, betaGal expression was detected in fetal hepatocytes and disappeared around birth. In adult mice, a CD8+ T-cell response to betaGal was observed after immunisation with betaGal adenovirus or plasmid DNA but not with betaGal protein or after retroviral infection. When betaGal was re-expressed in adult hepatocytes, immunisation with betaGal adenovirus triggered T-cell mediated elimination of betaGal-expressing hepatocytes. However, the response was weaker than in AFP/betaGal animals in which betaGal was only present around birth. CONCLUSIONS: In AFP/betaGal mice, betaGal is a fetal liver self-antigen. Interestingly, the basal tolerance to betaGal displayed by these animals is increased during liver re-expression of the self-antigen in adulthood. Adenoviral immunisation allows complete elimination of betaGal-expressing hepatocytes in spite of this increased peripheral tolerance. These results highlight the importance of tolerance against self-antigens and validate the AFP/betaGal mice as a good background to test immunotherapy strategies in hepatocarcinogenesis models.
Assuntos
Autoantígenos/uso terapêutico , Imunoterapia/métodos , Fígado/embriologia , beta-Galactosidase/imunologia , beta-Galactosidase/uso terapêutico , Adenoviridae/genética , Envelhecimento/metabolismo , Animais , Formação de Anticorpos , Linfócitos T CD8-Positivos/imunologia , DNA/uso terapêutico , Feto/metabolismo , Vetores Genéticos , Hepatócitos/metabolismo , Imunização , Fígado/patologia , Camundongos , Camundongos Transgênicos , Plasmídeos/uso terapêutico , Regiões Promotoras Genéticas , alfa-Fetoproteínas/genética , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Cytotoxic immune elimination of transduced hepatocytes may limit gene therapy for inherited liver diseases. Using beta-galactosidase as a marker gene, we studied whether creation of mixed beta-galactosidase molecular hematopoietic chimerism could induce tolerance to beta-galactosidase-transduced hepatocytes. METHODS: Molecular hematopoietic chimerism was established in irradiated recipient mice by transplantation of either a mixture of wild-type and beta-galactosidase-transgenic bone marrow or autologous bone marrow stem cells that were transduced with beta-galactosidase lentiviral vectors. After transplantation, mice were hepatectomized and injected with beta-galactosidase recombinant retroviruses to transduce regenerating hepatocytes. We monitored the presence of beta-galactosidase-expressing hepatocytes as well as the appearance of anti-beta-galactosidase antibodies during the time. RESULTS: In control animals, anti-beta-galactosidase antibodies and cytotoxic T-lymphocyte (CTL) response developed as early as 3 weeks after gene transfer. Transduced hepatocytes disappeared concomitantly. In bone marrow transplanted mice, tolerance could be observed in a significant proportion of animals. Tolerance resulted in permanent liver transgene expression and was absent unless a chimerism above 1% was achieved, demonstrating a threshold effect. CONCLUSIONS: Creation of a molecular hematopoietic chimerism can result in transgene tolerance and evade immune rejection of retrovirally transduced hepatocytes. This strategy may be useful for hepatic inherited diseases in which the transgene product behaves as a non-self protein.
Assuntos
Quimerismo , Hepatócitos/metabolismo , Lentivirus/genética , Transgenes , Animais , Transplante de Medula Óssea , Células Cultivadas , Galactosidases/genética , Galactosidases/imunologia , Galactosidases/metabolismo , Expressão Gênica , Sistema Hematopoético , Lentivirus/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Transdução GenéticaRESUMO
Crigler-Najjar type 1 disease (CN-1) is a genetic disorder characterized by high levels of unconjugated bilirubin due to the absence of hepatic UDPglucuronosyltransferase (UGT1) activity. Here we show that in vivo neonatal hepatocyte transduction with a lentiviral vector expressing the defective enzyme resulted in long-term correction in Gunn rats, a model of CN-1. Lentiviral vectors harboring the human UGT1 cDNA (approved symbol UGT1A1) under the control of a liver-specific transthyretin promoter were produced. Two-day-old Gunn rats were injected with 50 microl of vector. Bilirubinemia was monitored at 6 weeks and monthly thereafter. At 6 weeks, bilirubinemia was completely normalized in treated animals, whereas it remained around 100 microM in control rats. The level of correction remained stable for up to 42 weeks. Large amounts of bilirubin conjugates were present in the bile of corrected animals. PCR and Western blots confirmed the presence and expression of UGT1 in liver. The estimated proportion of transduced hepatocytes was 40% and transduced cells were not detected in extrahepatic tissues except bone marrow in some animals. This work represents the first demonstration of a complete and permanent correction of hyperbilirubinemia in Gunn rats using lentiviral vectors.