RESUMEN
The human parvovirus adeno-associated virus type 2 (AAV-2) possesses many features that make it an attractive vector for gene delivery in vivo. However, its broad host range may limit its usefulness and effectivity in several gene therapy applications in which transgene expression needs to be limited to a specific organ or cell type. In this study, we explored the possibility of directing recombinant AAV-2 transduction by incorporating targeting peptides previously isolated by in vivo phage display. Two putative loops within the AAV-2 capsid were examined as sites for incorporation of peptides. We tested the effects of deleting these loops and different strategies for the incorporation of several targeting peptides. The tumor-targeting sequence NGRAHA and a Myc epitope control were incorporated either as insertions or as replacements of the original capsid sequence. Viruses were assessed for packaging, accessibility of incorporated peptides, heparin binding, and transduction in a range of cell lines. Whereas recombinant viruses containing mutant capsid proteins were produced efficiently, transduction of several cell lines was significantly impaired for most modifications. However, certain mutants containing the peptide motif NGR, which binds CD13 (a receptor expressed in angiogenic vasculature and in many tumor cell lines), displayed an altered tropism toward cells expressing this receptor. Based on this work and previous studies, possible strategies for achieving in vivo targeting of recombinant AAV-2 are discussed.
Asunto(s)
Antígenos CD13/genética , Cápside/genética , Dependovirus/genética , Oligopéptidos/genética , Transducción Genética , Secuencia de Aminoácidos , Western Blotting , Antígenos CD13/metabolismo , ADN/metabolismo , Cartilla de ADN/química , Genes myc/genética , Proteínas Fluorescentes Verdes , Heparina/metabolismo , Humanos , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Mutación , Parvovirus/química , Parvovirus/genética , Reacción en Cadena de la Polimerasa , Receptores Virales/metabolismo , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
3'-End-capped, 20-mer antisense oligodeoxynucleotides (AS-ODN) protected with 2'-O-methyl (Me) or phosphorothioate (PS) substitutions were targeted to acetylcholinesterase (AChE) mRNA and studied in PC12 cells. Me-modified AS-ODN suppressed AChE activity up to 50% at concentrations of 0.02-100 nM. PS-ODN was effective at 1-100 nM. Both AS-ODN displayed progressively decreased efficacy above 10 nM. In situ hybridization and confocal microscopy demonstrated dose-dependent decreases, then increases, in AChE mRNA. Moreover, labeling at nuclear foci suggested facilitated transcription or stabilization of AChE mRNA or both under AS-ODN. Intracellular concentrations of biotinylated oligonucleotide equaled those of target mRNA at extracellular concentrations of 0.02 nM yet increased only 6-fold at 1 microM ODN. Above 50 nM, sequence-independent swelling of cellular, but not nuclear, volume was observed. Our findings demonstrate suppressed AChE expression using extremely low concentrations of AS-ODN and attribute reduced efficacy at higher concentrations to complex host cell feedback responses.
Asunto(s)
Acetilcolinesterasa/genética , Oligodesoxirribonucleótidos Antisentido/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Expresión Génica/efectos de los fármacos , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Células PC12 , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Gene therapy would be considerably more effective if vectors could be targeted to specific organs or tissues after systemic administration. We previously developed an in vivo selection system to isolate organ- and tumor-homing peptides from phage display peptide libraries. The peptides isolated by this approach bind to receptors expressed in vascular endothelia. We describe here the development of molecular adaptors to target adenoviral gene therapy vectors to selective vascular "addresses." The adaptor design consists of an organhoming peptide conjugated to an adenovirus-binding moiety. We isolated and characterized several monoclonal antibodies that bind to adenovirus type 5 (Ad5). Two of the antibodies neutralized Ad5 infection. We linked the Fab fragments of one of these antibodies to a synthetic lung-homing peptide (CGFECVRQCPERC or GFE-1 peptide) and tested the ability of the resulting bispecific conjugate to retarget Ad5. Cells that express the receptor for the GFE-1 peptide and are resistant to Ad5 infection were sensitized to recombinant Ad5 vectors in the presence of the Fab-GFE adaptor. Our findings indicate that selective gene therapy delivery may be developed on the basis of our vascular targeting technology.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos , Adenoviridae/genética , Adenoviridae/inmunología , Adenoviridae/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Western Blotting , Línea Celular , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Células HeLa , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Biblioteca de Péptidos , Pruebas de Precipitina , Células Tumorales CultivadasRESUMEN
The 34-kDa product of adenovirus E4 region open reading frame 6 (E4orf6) dramatically enhances transduction by recombinant adeno-associated virus vectors (rAAV). This is achieved by promoting the conversion of incoming single-stranded viral genomes into transcriptionally competent duplex molecules. The molecular mechanism for enhancing second-strand synthesis is not fully understood. In this study, we analyzed the cellular consequences of E4orf6 expression and the requirements for efficient rAAV transduction mediated by E4orf6. Expression of E4orf6 in 293 cells led to an inhibition of cell cycle progression and an accumulation of cells in S phase. This was preceded by specific degradation of cyclin A and p53, while the levels of other proteins involved in cell cycle control remained unchanged. In addition, the kinase activity of cdc2 was inhibited. We further showed that p53 expression is not necessary or inhibitory for augmentation of rAAV transduction by E4orf6. However, overexpression of cyclin A inhibited E4orf6-mediated enhancement of rAAV transduction. A cyclin A mutant incapable of recruiting protein substrates for cdk2 was unable to inhibit E4orf6-mediated augmentation. In addition, we created an E4orf6 mutant that is selectively defective in rAAV augmentation of transduction. Based on these findings, we suggest that cyclin A degradation represents a viral mechanism to disrupt cell cycle progression, resulting in enhanced viral transduction. Understanding the cellular pathways used during transduction will increase the utility of rAAV vectors in a wide range of gene therapy applications.
Asunto(s)
Proteínas E4 de Adenovirus/metabolismo , Transformación Celular Viral , Ciclina A/fisiología , Dependovirus , Proteínas E4 de Adenovirus/antagonistas & inhibidores , Proteínas E4 de Adenovirus/genética , Sitios de Unión , Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Línea Celular , Ciclina A/biosíntesis , Ciclina A/genética , Dependovirus/genética , Expresión Génica , Humanos , Recombinación Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
To explore role(s) of acetylcholinesterase (AChE) in functioning and diseased photoreceptors, we studied normal (rd/+) and degenerating (rd/rd) murine retinas. All retinal neurons, expressed AChEmRNA throughout fetal development. AChE and c-Fos mRNAs peaked at post-natal days 10-12, when apoptosis of rd/rd photoreceptors begins. Moreover, c-Fos and AChEmRNA were co-overexpressed in rd/rd mice producing transgenic human (h), and host (m) AChE, but not in rd/+ mice. However, mAChE overexpression also occurred in transgenics expressing human serum albumin. Drastic variations in AChE catalytic activity were ineffective during development. Neither transgenic excess nor diisopropylfluorophosphonate (DFP) inhibition (80%) affected the rd phenotype; nor did DFP exposure induce photoreceptor degeneration or affect other key cholinergic proteins in rd/+ mice, unlike reports of adult mice and despite massive induction under DFP of c-Fos70 years). Therefore, the extreme retinal sensitivity to AChE modulation may reflect non-catalytic function(s) of AChE in adult photoreceptors. These findings exclude AChE as causing the rd phenotype, suggest that its primary function(s) in mammalian retinal development are non-catalytic ones and indicate special role(s) for the AChE protein in adult photoreceptors.
Asunto(s)
Acetilcolinesterasa/genética , Regulación Enzimológica de la Expresión Génica , Células Fotorreceptoras de Vertebrados/fisiología , Degeneración Retiniana/enzimología , Adulto , Envejecimiento/genética , Animales , Catálisis , Inhibidores de la Colinesterasa/farmacología , ADN Nucleotidilexotransferasa/metabolismo , Humanos , Ratones , Ratones Transgénicos , FenotipoRESUMEN
Our growing understanding of the role that unfavorable patterns of gene expression play in the etiology of neurodegenerative disease emphasizes the need for strategies to selectively block the biosynthesis of harmful proteins in the brain. Antisense technologies are ideally suited to this purpose. Tailor-designed to target specific RNA, antisense oligonucleotides and ribozymes offer tools to suppress the production of proteins mediating neurodegeneration. Although technical limitations must still be overcome, the antisense approach represents a novel and exciting strategy for intervention in diseases of the central nervous system.
Asunto(s)
Sistemas de Liberación de Medicamentos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/uso terapéutico , ARN Catalítico/administración & dosificación , ARN Catalítico/uso terapéutico , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , ARN Catalítico/metabolismoRESUMEN
Accumulated evidence attributes noncatalytic morphogenic activitie(s) to acetylcholinesterase (AChE). Despite sequence homologies, functional overlaps between AChE and catalytically inactive AChE-like cell surface adhesion proteins have been demonstrated only for the Drosophila protein neurotactin. Furthermore, no mechanism had been proposed to enable signal transduction by AChE, an extracellular enzyme. Here, we report impaired neurite outgrowth and loss of neurexin Ialpha mRNA under antisense suppression of AChE in PC12 cells (AS-ACHE cells). Neurite growth was partially rescued by addition of recombinant AChE to the solid substrate or by transfection with various catalytically active and inactive AChE variants. Moreover, overexpression of the homologous neurexin I ligand, neuroligin-1, restored both neurite extension and expression of neurexin Ialpha. Differential PCR display revealed expression of a novel gene, nitzin, in AS-ACHE cells. Nitzin displays 42% homology to the band 4.1 protein superfamily capable of linking integral membrane proteins to the cytoskeleton. Nitzin mRNA is high throughout the developing nervous system, is partially colocalized with AChE, and increases in rescued AS-ACHE cells. Our findings demonstrate redundant neurite growth-promoting activities for AChE and neuroligin and implicate interactions of AChE-like proteins and neurexins as potential mediators of cytoarchitectural changes supporting neuritogenesis.
Asunto(s)
Acetilcolinesterasa/fisiología , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuritas/fisiología , Neuronas/fisiología , Neuronas/ultraestructura , Acetilcolinesterasa/farmacología , Secuencia de Aminoácidos , Animales , Moléculas de Adhesión Celular Neuronal , Técnicas de Transferencia de Gen , Glicoproteínas , Datos de Secuencia Molecular , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Neuropéptidos , Oligonucleótidos Antisentido/farmacología , Células PC12 , RatasRESUMEN
To investigate the effect of neuronal differentiation on the capacity of antisense oligonucleotides (AS-ODNs) to suppress the production of acetylcholinesterase (AChE) in rat pheochromocytoma cells, we tested seven 3'-phosphorothioated AS-ODNs targeted to ACHEmRNA and two control ODNs. Three different administration protocols were used: oligonucleotides were added at 1 microM for 24 hours to nondifferentiated PC12 cells, together with nerve growth factor (NGF) or 24 hours following NGF-induced cholinergic differentiation. The content of free thiol groups in lysed cells was measured to evaluate cell number, therefore, survival, and the rate of acetylthiocholine hydrolysis was the measure of AChE activity. Among nondifferentiated cells, over 95% survived treatment with 8 of 9 of the ODNs. Moreover, two AS-ODN suppressed AChE activity in non-differentiated PC12 cells by 16%-20% as compared with 10% suppression by control ODNs (P < or = 0.01). When added concurrently with NGF, one other AS-ODN suppressed AChE activity significantly better (28%) than the control ODNs (16%). Moreover, when added following NGF treatment, which induced a significant increase in AChE activity, four different AS-ODNs but not the control ODNs suppressed 20%-35% of the enhanced AChE activity (p < or = 0.01). Reduced levels of AChE mRNA but no difference in actin mRNA levels were observed by following the kinetics of RT-PCR amplification in differentiated PC12 cells treated with these four AS-ODNs, as compared with control cells. Our findings demonstrate a differentiation-related increase in the susceptibility of PC12 cells to inhibition by specific AS-ODNs, suggesting the use of this model system to select AS-ODNs for suppression of AChE levels in the treatment of neurodegenerative diseases associated with cholinergic malfunction.
Asunto(s)
Acetilcolinesterasa/biosíntesis , Encéfalo/citología , Inhibidores de la Colinesterasa , Neuronas/citología , Oligonucleótidos Antisentido/farmacología , Feocromocitoma/enzimología , Transcripción Genética/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Actinas/biosíntesis , Neoplasias de las Glándulas Suprarrenales/enzimología , Animales , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Cinética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Células PC12 , ARN Mensajero/biosíntesis , Ratas , TionucleótidosRESUMEN
Several approaches have been developed for genetic modulations of receptor expression. These initiated with gene cloning and heterologous expression in microinjected Xenopus oocytes, and proceeded through transgenic expression and genomic disruption of receptor genes in mice. In addition, antisense treatments have reduced receptor levels in a transient, reversible manner. Integration of foreign DNA with host genomic sequences yields both cis- and trans-acting responses. These may depend on the DNA integration site, host cells condition and most importantly, the affected signal transduction circuit. For example, acetylcholinesterase (AChE) overexpression in microinjected Xenopus tadpoles has been shown to upregulate alpha-bungarotoxin binding levels, indicating trans-acting control conferring overproduction of muscle nicotinic acetylcholine receptors. In transgenic mice expressing human AChE, the hypothermic response to oxotremorine was suppressed, reflecting modified levels of brain muscarinic receptors. To dissociate the feedback processes occurring in transfected cells from responses related to DNA integration, we examined the endogenous expression of the alpha 7 neuronal nicotinic acetylcholine receptor in PC12 cells transfected with DNA vectors carrying alternative splicing variants of human AChE mRNA. Our findings demonstrate suppression of alpha 7 receptor levels associated with the accumulation of foreign DNA in the transfected cells. Acetylcholine receptor levels thus depend on multiple elements, each of which should be considered when genetic interventions are employed.
Asunto(s)
Receptores Colinérgicos/genética , Activación Transcripcional , Acetilcolinesterasa/metabolismo , Animales , Animales Modificados Genéticamente , Humanos , Mutagénesis Sitio-Dirigida , Oligonucleótidos Antisentido/farmacología , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismoRESUMEN
Here, we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) but not casein kinase II or protein kinase C phosphorylates recombinant human acetylcholinesterase (AChE) in vitro. This enhances acetylthiocholine hydrolysis up to 10-fold as compared to untreated AChE, while leaving unaffected the enzyme's affinity for this substrate and for various active and peripheral site inhibitors. Alkaline phosphatase treatment enhanced the electrophoretic migration, under denaturing conditions, of part of the AChE proteins isolated from various mammalian sources and raised the isoelectric point of some of the treated AChE molecules, indicating that part of the AChE molecules are also phosphorylated in vivo. Enhancement of acetylthiocholine hydrolysis also occurred with Torpedo AChE, which has no consensus motif for PKA phosphorylation. Further, mutating the single PKA site in human AChE (threonine-249) did not prevent this enhancement, suggesting that in both cases it was due to phosphorylation at non-consensus sites. In vivo suppression of the acetylcholine hydrolyzing activity of AChE and consequent impairment in cholinergic neurotransmission occur under exposure to both natural and pharmacological compounds, including organophosphate and carbamate insecticides and chemical warfare agents. Phosphorylation of AChE may possibly offer a rapid feedback mechanism that can compensate for impairments in cholinergic neurotransmission, modulating the hydrolytic activity of this enzyme and enabling acetylcholine hydrolysis to proceed under such challenges.
Asunto(s)
Acetilcolina/metabolismo , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Conformación Proteica , Acetiltiocolina/metabolismo , Animales , Encéfalo/enzimología , Bovinos , Clonación Molecular , Secuencia de Consenso , Proteínas Quinasas Dependientes de AMP Cíclico/química , Eritrocitos/enzimología , Escherichia coli , Humanos , Hidrólisis , Cinética , Sustancias Macromoleculares , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina , TreoninaRESUMEN
To study the mechanisms underlying cholinotoxic brain damage, we examined ethylcholine aziridinium (AF64A) effects on cholinesterase genes. In vitro, AF64A hardly affected cholinesterase activities yet inhibited transcription of the G,C-rich AChE DNA encoding acetylcholinesterase (AChE) more than the A,T-rich butyrylcholinesterase (BChE) DNA. In vivo, intracerebroventricular injection of 2 nmol of AF64A decreased AChE mRNA in striatum and septum by 3- and 25-fold by day 7, with no change in BChE mRNA or AChE activity. In contrast, hippocampal AChE mRNA increased 10-fold by day 7 and BChE mRNA and AChE activity decreased 2-fold. By day 60 post-treatment, both AChE mRNA and AChE levels returned to normal in all regions except hippocampus, where AChE activity and BChE mRNA were decreased by 2-fold. Moreover, differential PCR displays revealed persistent induction, specific to the hippocampus of treated rats, of several unidentified G,C-rich transcripts, suggesting particular responsiveness of hippocampal G,C-rich genes to cholinotoxicity.
Asunto(s)
Acetilcolinesterasa/biosíntesis , Aziridinas/farmacología , Encéfalo/enzimología , Butirilcolinesterasa/biosíntesis , Ventrículos Cerebrales/fisiología , Colina O-Acetiltransferasa/metabolismo , Colina/análogos & derivados , Expresión Génica/efectos de los fármacos , Bloqueantes Neuromusculares/farmacología , Análisis de Varianza , Animales , Aziridinas/administración & dosificación , Secuencia de Bases , Ventrículos Cerebrales/efectos de los fármacos , Colina/administración & dosificación , Colina/farmacología , Cartilla de ADN , Inyecciones Intraventriculares , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
To examine the role of acetylcholinesterase (EC 3.1.1.7) in hematopoietic cell proliferation and differentiation, we administered a 15-mer phosphorothioate oligonucleotide, antisense to the corresponding ACHE gene (AS-ACHE), to primary mouse bone marrow cultures. Within 2 hr of AS-ACHE addition to the culture, ACHE mRNA levels dropped by approximately 90%, as compared with those in cells treated with the "sense" oligomer, S-ACHE. Four days after AS-ACHE treatment, ACHE mRNA increased to levels 10-fold higher than in S-ACHE cultures or in fresh bone marrow. At this later time point, differential PCR display revealed significant differences between cellular mRNA transcripts in bone marrow and those in AS-ACHE- or S-ACHE-treated cultures. These oligonucleotide-triggered effects underlay considerable alterations at the cellular level: AS-ACHE but not S-ACHE increased cell counts, reflecting enhanced proliferation. In the presence of erythropoietin it also enhanced colony counts, reflecting expansion of progenitors. AS-ACHE further suppressed apoptosis-related fragmentation of cellular DNA in the progeny cells, and it diverted hematopoiesis toward production of primitive blasts and macrophages in a dose-dependent manner promoted by erythropoietin. These findings suggest that the hematopoietic role of acetylcholinesterase, anticipated to be inverse to the observed antisense effects, is to reduce proliferation of the multipotent stem cells committed to erythropoiesis and megakaryocytopoiesis and macrophage production and to promote apoptosis in their progeny. Moreover, these findings may explain the tumorigenic association of perturbations in ACHE gene expression with leukemia.