RESUMEN
Cyclic neutropenia occurs in humans and gray collie dogs, is characterized by recurrent neutropenia, and is treated by daily injections of recombinant granulocyte colony-stimulating factor (G-CSF). After showing that canine recombinant G-CSF increased neutrophil counts in an affected dog, we administered intramuscularly 2 x 10(9) infectious units (IU) of a lentiviral vector encoding canine G-CSF cDNA. Elevated, therapeutic neutrophil production was obtained for nearly 18 months. Lentiviral vector treatment provided a mean neutrophil count of 29,230 +/- 12,930 cells/microl, which was significantly increased over both the pretreatment value (5,240 +/- 4,800 cells/microl; p < 0.0001) and the neutrophil count during G-CSF administration (17,820 +/- 11,100 cells/microl; p < 0.0001). By systemic infusion of recombinant G-CSF to normal dogs we estimated that 2 x 10(9) IU of lentivirus delivered 3.5 microg of G-CSF per kilogram per day. After lentiviral vector treatment the gray collie gained weight, showed no clinical signs of infection and fever, and no longer needed housing in a pathogen-free environment. Genomic DNA harvested from muscle at the injection sites was positive for provirus, whereas gonad, lung, spleen, heart, liver, kidney, and noninjected muscle samples were negative. These studies show that an adult animal is responsive long-term to lentivirus-mediated G-CSF delivery, suggesting this approach may be applied for treatment of adult patients with cyclic and other neutropenias.
Asunto(s)
Enfermedades de los Perros/terapia , Terapia Genética , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos/genética , Lentivirus , Neutropenia/veterinaria , Animales , Enfermedades de los Perros/genética , Perros , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Neutropenia/genética , Neutropenia/terapia , Proteínas Recombinantes , Factores de TiempoRESUMEN
Smooth muscle alpha-actin (SMA) is a marker for the contractile, non-proliferative phenotype of adult smooth muscle cells (SMCs). Upon arterial injury, expression of SMA and other structural proteins decreases and SMCs acquire a pro-migratory and proliferative phenotype. To what extent SMA regulates migration and proliferation of SMCs is unclear and putative signaling pathways involved remain to be elucidated. Here, we used lentiviral-mediated gene transfer and siRNA technology to manipulate expression of SMA in carotid mouse SMCs and studied effects of SMA. Overexpression of SMA results in decreased proliferation and migration and blunts serum-induced activation of the small GTPase Rac, but not RhoA. All inhibitory effects of SMA are rescued by expression of a constitutively active Rac1 mutant (V12rac1). Moreover, reduction of SMA expression by siRNA technology results in an increased activation of Rac. Taken together, this study identifies Rac1 as a downstream target for SMA to inhibit SMC proliferation and migration.
Asunto(s)
Actinas/metabolismo , Movimiento Celular , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Animales , Proliferación Celular , Activación Enzimática , Adhesiones Focales/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación/genética , Suero/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Recombinant erythropoietin (EPO) is widely administered for long-term treatment of anemia associated with renal failure and other chronic diseases. The ability to deliver EPO by gene therapy would have clinical and economic benefit. We compared autologous and allogeneic transduced primary vascular smooth muscle cells for their ability to provide sustained EPO gene expression when encapsulated in TheraCyte devices implanted subcutaneously (SQ) or intraperitoneally (IP) in rats. Cells were transduced with retrovirus vector LrEpSN encoding rat EPO cDNA. Rats that received either autologous or allogeneic transduced cells showed elevated hematocrits (HCTs) ranging from 50 to 79% that were sustained for more than 12 months. The HCT of control rats remained at baseline (45.8%). Rats that received second SQ implants of either autologous or allogeneic cells showed elevations in hematocrit that were sustained for up to 12 months, suggesting the absence of immunological responses to transduced cells or implant material. All experimental groups had statistically significant elevated HCT (p < 0.001) when compared with controls. Both SQ and IP implantation were equally effective in delivering EPO long term. There were no significant differences in white blood cell (WBC) or platelet (PLT) values between treated and control animals. Implantation of TheraCyte devices was well tolerated and histological evaluation of the devices up to 12 months after surgery revealed a high degree of vascularization and no evidence of host immune response. TheraCyte devices offer a simple and safe gene delivery system that provides sustained therapeutic gene expression, permit removal and implantation of new devices, and do not require immunosuppression of the host.
Asunto(s)
Eritropoyetina/biosíntesis , Eritropoyetina/genética , Expresión Génica , Terapia Genética/métodos , Anemia/terapia , Animales , Plaquetas/metabolismo , Células Cultivadas , ADN Complementario/metabolismo , Vectores Genéticos , Hematócrito , Leucocitos/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Ratas , Ratas Endogámicas F344 , Ratas Wistar , Retroviridae/genética , Factores de TiempoRESUMEN
Retroviral vectors are commonly used in ex vivo gene therapy protocols. The structure of vectors basically consists of one gene of interest and a selectable marker gene. Fast selection without damaging cells is a critical step for ex vivo gene therapy protocols. Blasticidin S deaminase isolated from Bacillus cereus has a neutralizing action on the highly toxic antibiotic blasticidin S (BS). A commercially available gene coding for blasticidin S deaminase (bsr) when used to construct retroviral vectors, LBSN and LNSB, provided very low levels of BS deaminase activity, precluding their routine use in gene transfer experiments. However, with the introduction of specific mutations into the bsr gene based on the Kozak consensus sequences and deletion of a 5' untranslated sequence to generate bsrm, we were able to construct a retroviral vector encoding resistance to high doses of BS (at least 16-fold above the usual lethal dose in NIH3T3 cells), showing that bsrm/BS may provide a useful system for selection of transduced mammalian cells.
Asunto(s)
Aminohidrolasas/genética , Farmacorresistencia Viral/genética , Marcadores Genéticos/genética , Vectores Genéticos/genética , Nucleósidos/farmacología , Retroviridae/genética , Transducción Genética/métodos , Células 3T3 , Animales , Terapia Genética/métodos , Ratones , Virus de la Leucemia Murina de Moloney/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Selección GenéticaRESUMEN
Human cyclic neutropenia, also referred to as cyclic hematopoiesis, is a rare disease characterized by periodic fluctuations in blood cell production by the bone marrow and a corresponding recurrent severe neutropenia every 19-21 days. This results in bacterial infections and shortened life expectancy. Platelets, monocytes, lymphocytes, and reticulocytes cycle with the same periodicity. It has been determined that the neutrophil elastase (NE) gene is mutated in all cases of human cyclic hematopoiesis. Currently, the only animal model for this disease is the grey collie dog, in which there is a strikingly similar periodic neutropenia every 12-14 days. Towards the validation of this animal model, we have cloned and sequenced the canine NE cDNA from a normal dog.
Asunto(s)
Elastasa de Leucocito/genética , Neutropenia/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/análisis , Modelos Animales de Enfermedad , Perros , Datos de Secuencia Molecular , Mutación , Neutropenia/sangre , Neutropenia/enzimología , Neutrófilos/metabolismoRESUMEN
Cyclic neutropenia occurs in humans and gray collie dogs, is characterized by recurrent neutropenia, and is treated by repeated injections of recombinant granulocyte colony-stimulating factor (rG-CSF). As dose escalation of lentivirus may be clinically necessary, we monitored the outcome of four sequential intramuscular injections of G-CSF-lentivirus (3 × 10(7) IU/kg body weight) to a normal dog and a gray collie. In the normal dog absolute neutrophil counts were significantly increased after each dose of virus, with mean levels of 27.75 ± 3.00, 31.50 ± 1.40, 35.05 ± 1.68, and 43.88 ± 2.94 × 10(3) cells/µl, respectively (p<0.001), and elevated neutrophil counts of 31.18 ± 7.81 × 10(3) cells/µl were maintained for more than 6 years with no adverse effects. A gray collie dog with a mean count of 1.94 ± 1.48 × 10(3) cells/µl received G-CSF-lentivirus and we observed sustained elevations in neutrophil levels for more than 5 months with a mean of 26.00 ± 11.00 × 10(3) cells/µl, significantly increased over the pretreatment level (p<0.001). After the second and third virus administrations mean neutrophil counts of 15.80 ± 6.14 and 11.52 ± 4.90 × 10(3) cells/µl were significantly reduced compared with cell counts after the first virus administration (p<0.001). However, after the fourth virus administration mean neutrophil counts of 15.21 ± 4.50 × 10(3) cells/µl were significantly increased compared with the previous administration (p<0.05). Throughout the nearly 3 years of virus administrations the dog gained weight, was healthy, and showed neutrophil counts significantly higher than pretreatment levels (p<0.001). These studies suggest that patients with cyclic and other neutropenias may be treated with escalating doses of G-CSF-lentivirus to obtain a desired therapeutic neutrophil count.
Asunto(s)
Enfermedades de los Perros/terapia , Vectores Genéticos/genética , Factor Estimulante de Colonias de Granulocitos/genética , Lentivirus/genética , Neutropenia/veterinaria , Animales , Enfermedades de los Perros/genética , Perros , Femenino , Terapia Genética , Vectores Genéticos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/metabolismo , Recuento de Leucocitos , Masculino , Neutropenia/genética , Neutropenia/terapiaRESUMEN
Immunoprotection of islets using bioisolator systems permits introduction of allogeneic cells to diabetic patients without the need for immunosuppression. Using TheraCyte immunoisolation devices, we investigated two rat models of type 1 diabetes mellitus (T1DM), BB rats and rats made diabetic by streptozotocin (STZ) treatment. We chose to implant islets after the onset of diabetes to mimic the probable treatment of children with T1DM as they are usually diagnosed after disease onset. We encapsulated 1000 rat islets and implanted them subcutaneously (SQ) into diabetic biobreeding (BB) rats and STZ-induced diabetic rats, defined as two or more consecutive days of blood glucose>350 mg/dl. Rats were monitored for weight and blood glucose. Untreated BB rats rapidly lost weight and were euthanized at >20% weight loss that occurred between 4 and 10 days from implantation. For period of 30-40 days following islet implantation weights of treated rats remained steady or increased. Rapid weight loss occurred after surgical removal of devices that contained insulin positive islets. STZ-treated rats that received encapsulated islets showed steady weight gain for up to 130 days, whereas untreated control rats showed steady weight loss that achieved >20% at around 55 days. Although islet implants did not normalize blood glucose, treated rats were apparently healthy and groomed normally. Autologous or allogeneic islets were equally effective in providing treatment. TheraCyte devices can sustain islets, protect allogeneic cells from immune attack and provide treatment for diabetic-mediated weight loss in both BB rats and STZ-induced diabetic rats.
Asunto(s)
Diabetes Mellitus Experimental/terapia , Trasplante de Islotes Pancreáticos , Animales , Peso Corporal , Femenino , Masculino , Implantación de Prótesis , Ratas , Estreptozocina , Trasplante HomólogoRESUMEN
BACKGROUND: Towards gene therapy treatment of patients with neutropenia, characterized by neutrophil counts < 500 cells/microl, we investigated the ability of lentivirus vectors to provide sustained granulocyte colony-stimulating factor (G-CSF) delivery and to permit transgene expression from a second virus administration in a preclinical rat model. METHODS: Rats were injected intramuscularly (IM) with 24 x 10(6) and 9 x 10(6) infectious units (IU) of a VSV-G-pseudotyped self-inactivating (SIN) lentivirus encoding rat G-CSF cDNA and containing cPPT and PRE elements. To determine the effectiveness of a second virus administration treated rats and a naïve rat received erythropoietin (EPO)-lentivirus IM. Rats were monitored for neutrophil and red blood cell production. Lentivirus antibodies were assayed by virus-neutralizing assay and ELISA. RESULTS: High and low dose virus administration increased neutrophil counts to 5660 +/- 930 cells/microl (mean +/- SD) and 4010 +/- 850 cells/microl, respectively, that were sustained for > 17 months and were significantly higher than controls counts of 1890 +/- 570 cells/microl (p< or =0.0002). Rats treated with EPO-virus produced significantly increased hematocrits (HCT) (63.1 +/- 4.3% vs. 46.0 +/- 3.2%, p < 0.0001) without effect on G-CSF-virus-mediated neutrophil production. Antivirus antibodies were not detectable at serum dilutions > or =1:10 by virus neutralization or ELISA. Lymphocytes and platelets were not significantly different between control and treated animals (p > 0.1). Only genomic DNA from muscle at injection sites was positive for provirus suggesting lack of virus spread. CONCLUSIONS: G-CSF-lentivirus administered IM provided elevated, sustained neutrophil counts that were unchanged by subsequent EPO-lentivirus administration. Significantly increased hematocrits were obtained following EPO-lentivirus delivery. These data support the treatment of patients with severe chronic neutropenia by dosed lentivirus delivery IM.
Asunto(s)
Eritropoyetina/genética , Eritropoyetina/metabolismo , Terapia Genética , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Lentivirus/genética , Neutrófilos/citología , Animales , Anticuerpos Antivirales/sangre , Recuento de Células Sanguíneas , ADN/metabolismo , Genoma/genética , Células HeLa , Hematócrito , Humanos , Masculino , Provirus/metabolismo , Ratas , Ratas Endogámicas F344RESUMEN
Cytidine deaminase (CDA) hydrolytically deaminates and irreversibly deactivates the chemotherapeutic agent cytosine arabinoside (Ara-C), a deoxycytidine analog used for treatment of acute leukemias and lymphomas. To determine if single nucleotide polymorphisms (SNPs) in the promoter region of CDA affected gene expression, we sequenced approximately 1.6 Kb upstream of the CDA translation initiation site and containing the proximal promoter of CDA. We identified 6 SNPs; -92A>G, -205C>G, -451C>T, -897C>A, -1075A>G and -1181G>A. Based on predicted changes in transcription factor binding sites, three SNPs (-92A>G, -451C>T and -897C>A) were chosen for further investigation. The five haplotypes segregating in the population were cloned into a luciferase expression plasmid, transfected into Cos-1 cells and reporter activity measured at 24 and 48 h. Four haplotypes showed an average expression which was 2.5-fold higher at 24 h (P<0.0001) and 3.3-fold higher at 48 h (P<0.0001) than the lowest expressing haplotype. When reanalyzed as single SNP genotypes, the differences in expression were significant, except for -897 C/A, at 24 h, but the magnitude of difference was reduced, suggesting that no single SNP completely accounts for the expression differences observed at the haplotype level. As predicted from the in vitro analysis, individuals homozygous for common haplotype (ACC/ACC) showed higher levels of CDA enzymatic activity as individuals heterozygous for the wild type and low expressing haplotype (ACC/ATC). As CDA promoter region haplotypes may influence Ara-C chemosensitivity, shown here in in vitro and in vivo studies, the clinical relevance of these findings should be examined.
Asunto(s)
Citidina Desaminasa/genética , Haplotipos , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Animales , Células COS , Chlorocebus aethiops , Regulación de la Expresión Génica , Humanos , Análisis de Secuencia de ADN , TransfecciónRESUMEN
BACKGROUND: Patients with severe chronic and cyclic neutropenia, characterized by neutrophil numbers <500 cells/microl, are treated daily with recombinant granulocyte colony-stimulating factor (G-CSF). As an alternative delivery approach we investigated the ability of lentivirus vectors to provide sustained G-CSF expression. METHODS: Fischer rats were injected intramuscularly (IM) with vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus pRRL-CMV-G-CSF-SIN that encoded rat G-CSF cDNA regulated by the human cytomegalovirus (CMV) promoter and incorporated a self-inactivating (SIN) construct in the 3' long terminal repeat (LTR). Control rats received normal saline or lentivirus encoding the enhanced green fluorescent protein (eGFP). Rats were serially monitored for blood cell production and tissues assayed for provirus distribution. RESULTS: Rats receiving a single IM injection of lentivirus exhibited elevated neutrophil counts for 14 months. Virus administration of 6 x 10(7) infectious units induced sustained levels of neutrophil production having a mean +/- standard deviation (SD) of 5650 +/- 900 cells/microl and rats that received a 10-fold lower dose of virus showed mean neutrophil counts of 3340 +/- 740 cells/microl. These were significantly higher than the mean of control animals receiving saline or control lentivirus (1,760 +/- 540 cells/microl, P < 0.0001). White blood cell (WBC) counts were significantly elevated in treated over control animals (P < 0.0001). Hematocrits (P > 0.3), lymphocytes (P > 0.2) and platelets (P > 0.1) were not significantly different between control and treated animals. Genomic DNA from muscle at the injection sites was positive for provirus, whereas lung, spleen, liver, kidney and non-injected muscle samples were all negative, suggesting lack of virus spread. CONCLUSIONS: These studies indicate that lentivirus vectors administered IM provide sustained, therapeutic levels of neutrophils and suggest this approach to treat patients with severe and cyclic neutropenia.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Lentivirus/genética , Neutropenia/terapia , Provirus/aislamiento & purificación , Animales , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Factor Estimulante de Colonias de Granulocitos/genética , Proteínas Fluorescentes Verdes/genética , Hematócrito , Recuento de Leucocitos , Neutrófilos/fisiología , Reacción en Cadena de la Polimerasa , Ratas , Ratas Endogámicas F344 , Virus de la Estomatitis Vesicular Indiana/genéticaRESUMEN
Cyclic neutropenia is a rare disease that occurs both in humans and gray collie dogs and is characterized by recurrent severe neutropenia leading to bacterial infections and shortened life expectancy. Daily injections of recombinant granulocyte colony-stimulating factor (rG-CSF) are effective in shortening the period of severe neutropenia and reducing infections. After demonstrating that rG-CSF induced elevated neutrophil production in an affected dog, cytokine administration was stopped and 109 infectious units (IUs) of a lentivirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) encoding canine G-CSF cDNA was administered intramuscularly. Serial blood cell counts showed elevated neutrophil production for longer than 17 months. Although neutrophil counts continued to cycle, the range at nadirs was from 3710 to 5300 cells/microL, well above the nadirs before lentivirus administration. After the injection of lentivirus, mean neutrophil counts +/- SD were 12 460 +/- 4240 cells/microL, significantly increased over both pretreatment values of 3040 +/- 2540 cells/microL(P <.0001) and neutrophil counts during G-CSF administration of 10 290 +/- 4860 cells/microL(P <.007). The changes in blood counts from lentivirus injection were associated with absence of clinical signs of infection and fever. The gray collie continued to gain weight and was no longer housed in a pathogen-free environment. Genomic DNA from muscle at injection sites was positive for provirus, whereas gonad, lung, spleen, heart, liver, kidney, leukocytes, and noninjected muscle samples were all negative for provirus. Thus, intramuscular administration of lentivirus encoding G-CSF provided sustained therapeutic levels of neutrophils, suggesting this approach may be applied for long-term treatment of patients with cyclic and other neutropenias.