RESUMO
When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, we isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G.C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G.C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.
Assuntos
DNA Viral/metabolismo , Dependovirus/genética , Endodesoxirribonucleases/isolamento & purificação , Células HeLa/enzimologia , Eletroforese em Gel de Ágar , Endodesoxirribonucleases/metabolismo , Humanos , Plasmídeos , Recombinação Genética , Especificidade por SubstratoRESUMO
For many metabolic diseases, early correction of the inherited deficiency is required to prevent long-term sequelae. We examined the ability of adeno-associated virus (AAV) to mediate efficient gene transfer during the neonatal period in mice with the lysosomal storage disease mucopolysaccharidosis type VII (MPS VII). Quadriceps of newborn MPS VII mice were injected with an AAV vector containing human beta-glucuronidase (GUSB) cDNA. High-level intramuscular GUSB expression was seen as early as 2 weeks of age, and persisted for at least 16 weeks with no reduction in activity. In addition, GUSB activity was detected in both liver and spleen at later time points. The level of GUSB activity resulted in a significant reduction in lysosomal storage in the liver and a minimal reduction in the spleen at 16 weeks. However, the temporal and spatial pattern of hepatic GUSB activity, coupled with the presence of GUSB cDNA in liver sections, suggests that hematogenous dissemination of virus at the time of injection led to gene transfer to hepatic cells. These results demonstrate that AAV vectors can successfully infect neonatal muscle and persist through the rapid growth phase following birth. However, GUSB secretion from an intramuscular source is inefficient, limiting the therapeutic efficacy of this approach.
Assuntos
Animais Recém-Nascidos , Terapia Genética , Glucuronidase/genética , Glucuronidase/metabolismo , Mucopolissacaridose VII/terapia , Fatores Etários , Animais , Southern Blotting , Dependovirus/genética , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos/administração & dosagem , Glicosaminoglicanos/química , Humanos , Injeções Intramusculares , Fígado/citologia , Fígado/enzimologia , Fígado/patologia , Lisossomos/química , Camundongos , Camundongos Mutantes , Mucopolissacaridose VII/patologia , Baço/citologia , Baço/enzimologia , beta-N-Acetil-Hexosaminidases/químicaRESUMO
Most obese animal models, whether associated with genetic, diet-induced, or age-related obesity, display pronounced leptin resistance, rendering leptin supplement therapy ineffective in treating obesity. Ciliary neurotrophic factor (CNTF) has been recently used to invoke leptin-like signaling pathways, thereby circumventing leptin resistance. In the current study, we characterize immediate and long-term molecular events in the hypothalamus of rats exposed to the sustained ectopic expression of leptin, CNTF, or leukemia inhibitory factor, another neurocytokine of IL-6 family, all delivered centrally via a viral vector. The respective transgene-encoded ligands induced similar but not identical metabolic responses as assessed by the reduction in body weight gain and changes in food intake. To define molecular mechanisms of weight-reducing and anorexigenic action of cytokines, we have analyzed the gene expression profiles of 1300 brain-specific genes in the hypothalami of normal rats subjected to the prolonged cytokine action for 10 wk. We present evidence that constitutive expression of cytokines in the brain induces changes in gene expression characteristic of chronic inflammation leading to either temporal weight reduction (CNTF) or severe cachexia (leukemia inhibitory factor). Our results convey a cautionary note regarding potential use of the tested cytokines in therapeutic applications.
Assuntos
Fator Neurotrófico Ciliar/fisiologia , Metabolismo Energético/fisiologia , Interleucina-6/fisiologia , Leptina/fisiologia , Animais , Peso Corporal/efeitos dos fármacos , Fator Neurotrófico Ciliar/genética , Fator Neurotrófico Ciliar/farmacologia , DNA , Dependovirus/genética , Ingestão de Alimentos/efeitos dos fármacos , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Interleucina-6/genética , Interleucina-6/farmacologia , Leptina/genética , Fator Inibidor de Leucemia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/normas , Ratos , Ratos Sprague-DawleyRESUMO
To test the suitability of simian virus 40 (SV40) DNA as a vector for inserting DNA segments into the chromosomes of mammalian cells, an EcoRI-A fragment of bacteriophage lambda DNA was covalently joined to a fragment of SV40 DNA and used to transform mouse cells in culture. Three independent, morphologically transformed clones were obtained that were positive for SV40 T-antigen by immunofluorescence staining. DNA from each transformant was examined by restriction enzyme analysis and found to contain both lambda and SV40 sequences. Co-migration of some fragments containing lambda and SV40 sequences following digestion of transformed cell DNA by each of four different restriction enzymes indicated that part of the retained lambda and SV40 DNA was linked in two of the three lines. In the third line, however, none of the restriction fragments had both lambda and SV40 sequences. Although the presence of non-integrated lambda DNA was not excluded, at least some of the lambda DNA appeared to be linked to host cell DNA. Results of digestion by EcoRI suggested that in some cases the transforming linear molecule had probably circularized prior to integration.
Assuntos
Bacteriófago lambda/genética , Transformação Celular Viral , DNA Recombinante , DNA Viral/genética , Vírus 40 dos Símios/genética , Animais , Linhagem Celular , DNA/genética , Rim , Camundongos , Hibridização de Ácido NucleicoRESUMO
A new reiterated variant of simian virus 40 (SV40; dl1142) has been constructed. It should be useful for the purpose of cloning foreign pieces of DNA in SV40 virions. Up to 80% of the SV40 genome has been made available for substitution with foreign DNA and the vector contains a number of unique (single-cut) restriction sites which will facilitate cloning. The 5' and 3' regions of both the SV40 early and late messenger RNAs are included in the vector. Prokaryotic DNA has been successfully cloned in the early region of the vector. The transcriptional properties of the recombinant have been studied, and it was found that both the vector and insert DNA are transcribed, mainly as non-adenylated RNA.
Assuntos
Vetores Genéticos , Plasmídeos , Recombinação Genética , Vírus 40 dos Símios/genética , Clonagem Molecular , Variação Genética , Fenótipo , Transcrição GênicaRESUMO
PstI restriction fragments of the colicin Ib (ColIb) plasmid have been cloned into the Apr gene of the pBR322 vector. Colicin-producing clones (Col+) all contained two common PstI (L and U) fragments, 2000 and 800 bp long, respectively. All of these colicin producers were found to permit the normal growth of bacteriophage T5; the presence of the whole ColIb plasmid causes an abortive T5 or BF23 phage infection. None of the other clones selected for their inability to propagate T5 produced colicin. The clones (Abi+) that permitted only an abortive infection by T5 contained a single restriction fragment, O (1600 bp), which was not found in any of the colicin producers. Likewise, the specific fragments (L and U) found in the Col+ clones were not found in the Abi+ clones. These data are very hard to reconcile with the hypothesis of a colicin-induced cell deterioration after T5 phage infection.
Assuntos
Colicinas/fisiologia , Desoxirribonucleases de Sítio Específico do Tipo II , Fagos T/crescimento & desenvolvimento , Plasmídeos de Bacteriocinas , Sequência de Bases , Membrana Celular/metabolismo , Clonagem Molecular , Colicinas/genética , Enzimas de Restrição do DNA , Eletroforese em Gel de Ágar , Escherichia coli/genéticaRESUMO
PURPOSE: One enzyme/prodrug combination that has the potential to be used in virally directed enzyme/prodrug therapy (VDEPT) is the obligate 2-electron reducing enzyme, DT-diaphorase (NQO1), with bioreductive agents such as EO9. The present studies were undertaken to determine if this enzyme, as well as the reporter molecule, green fluorescent protein (GFP), could be expressed from a single dicistronic unit under control of the CMV promoter in an adeno-associated virus (AAV) background. METHODS: The human ovarian tumor cell line, SAU, and the mouse sarcoma cell line, KHT/iv, were studied due to their low level of NQO1 expression. These cells were transfected with pTRUF3-NQO1 using a liposome-mediated protocol. RESULTS: The results indicate that this construct has the ability to increase the total protein level of NQO1 by 66-fold in SAU and 102-fold in KHT/iv after 24 h. Furthermore, the level of NQO1 activity in SAU increased from undetectable levels to approximately 200 nmol/min/mg, and the NQO1 activity in KHT/iv increased approximately 10-fold following transfection. Expression of the GFP reporter was readily detectable in both cell types using FACS analysis. CONCLUSIONS: Taken together, these results indicate that this proviral AAV vector plasmid will allow for the production of a recombinant AAV, which can coordinately express the enzyme NQO1 and the GFP reporter for use in vivo in VDEPT studies with various bioreductive agents which are substrates for NQO1.
Assuntos
Vetores Genéticos/uso terapêutico , Proteínas Luminescentes/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Transfecção/métodos , Animais , Dependovirus , Feminino , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , Neoplasias Ovarianas/metabolismo , Sarcoma Experimental/metabolismo , Células Tumorais CultivadasRESUMO
Mucopolysaccharidosis type VII (MPS VII) is caused by a deficiency in the lysosomal enzyme beta-glucuronidase resulting in the accumulation of undegraded glycosaminoglycans in many tissues. A murine model of MPS VII shares many of the clinical, biochemical and histopathological features of human MPS VII and has provided an opportunity to study novel therapeutic approaches in a system with a uniform genetic background. Retroviral mediated gene therapy directed to the hematopoietic system or to artificial neo-organs resulted in low levels of enzyme in several tissues and reduced lysosomal storage in the liver and spleen. Partial correction of the disease in the eye was observed following an intravitreal injection of recombinant adenovirus. Neither retroviral nor adenoviral mediated gene transfer techniques resulted in a systemic reduction of lysosomal storage. Here we discuss several novel gene transfer approaches designed to increase the systemic levels of beta-glucuronidase in the MPS VII mouse.
Assuntos
Modelos Animais de Doenças , Terapia Genética , Mucopolissacaridose VII/veterinária , Doenças dos Roedores/terapia , Adenoviridae/genética , Animais , Transplante de Medula Óssea , Vetores Genéticos , Glucuronidase/metabolismo , Hematopoese , Camundongos , Mucopolissacaridose VII/patologia , Mucopolissacaridose VII/terapiaRESUMO
Trophic factor gene therapy may provide a rational treatment strategy for neurodegenerative disease. Recombinant adeno-associated virus vectors, incorporating a neuron-specific promoter driving bicistronic expression of green fluorescent protein and either nerve growth factor or brain-derived neurotrophic factor, transduced 10,000-15,000 neurons in the medial septum for periods of at least six months. Both cholinergic and non-cholinergic neurons expressed green fluorescent protein. Nerve growth factor and brain-derived neurotrophic factor vectors produced up to 50% increases in immunohistochemical detection of the acetylcholine-synthesizing enzyme in septal neurons ipsilateral to the injection. Increased levels of this enzyme, choline acetyltransferase, persisted for six months with the brain-derived neurotrophic factor vector. The nerve growth factor vector increased Trk receptor immunoreactivity in a volume of brain exceeding that of the transduced cells. Counterstaining for the neuronal marker, NeuN, or Nissl substance did not reveal any vector toxicity at any time-point. It therefore appears that the lasting effects of vector-mediated trophic factor gene transfer will offer a new approach for modulating septal cholinergic transmission and Trk receptor activity.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Colina O-Acetiltransferase/metabolismo , Fatores de Crescimento Neural/farmacologia , Prosencéfalo/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Dependovirus/genética , Expressão Gênica/fisiologia , Vetores Genéticos , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Fatores de Crescimento Neural/genética , Ratos , Ratos Sprague-Dawley , Receptor do Fator Neutrófico Ciliar , Recombinação Genética , Fatores de Tempo , Transgenes/genéticaRESUMO
OBJECTIVE: To determine the foci and duration of protein expression following virus-mediated gene transfer to the optic nerve. METHODS: A cytomegalovirus (CMV) promoter was linked to a lacZ-SV40 polyA reporter gene or a humanized green fluorescent protein (hgfp) reporter gene, then inserted into a bacterial plasmid containing adeno-associated virus (AAV) terminal repeat sequences. The CMV-lacZ or the CMV-hgfp construct were injected into the vitreous cavity of strain-13 guinea pigs. Controls consisted of eyes injected with AAV without the promoter and reporter elements or eyes that received no injections. The eyes and optic nerves were processed for beta-galactosidase immunohistochemistry and hgfp fluorescence analyses. Cellular transduction at the messenger RNA (mRNA) level was evaluated by in situ reverse transcription-polymerase chain reaction. RESULTS: Weekly fundus photography, done for 1 month, documented the absence of any ocular abnormality due to the viral injections. No in vivo hgfp fluorescence of the retina was visualized. Beta-galactosidase histochemical analysis of eye cups that received the lacZ gene construct showed blue lacZ staining of the optic nerve head at 2 weeks. Light microscopy revealed the blue beta-galactosidase reaction product in fibers, glial cells, and blood vessels of the optic nerve head and retrobulbar nerve. Histochemistry showed absence of beta-galactosidase in the optic nerve at 3 to 12 months, but immunochemistry showed the persistence of beta-galactosidase in fibers, glial cells, and blood vessels as late as 1 year after a single ocular injection. In the retina, histochemical staining showed evidence of lacZ at 3 months, but not later. In situ reverse transcription-polymerase chain reaction revealed brown lacZ mRNA reaction product in ganglion cells of the retina. Control eyes that received AAV without the promoter and reporter elements and the eyes that received no viral injections and were processed for beta-galactosidase showed no reporter gene expression in any ocular tissue or cell type. CONCLUSIONS: Viral-mediated gene transfer can be successfully accomplished in the optic nerve. Further evaluation is needed to determine whether the level of protein expression at 1 year after injection, which is clearly reduced relative to shorter postinjection time, is sufficient for therapeutic purposes. CLINICAL RELEVANCE: We have previously shown that gene therapy with catalase suppressed experimental optic neuritis at 1 month after injection. Viral-mediated gene transfer may be a powerful technique for the treatment of optic neuropathies, particularly for recurrences of optic neuritis, if long-term expression of transduced protein can be demonstrated in the optic nerve.
Assuntos
Dependovirus/genética , Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter , Nervo Óptico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Citomegalovirus/genética , Proteínas de Fluorescência Verde , Cobaias , Técnicas Imunoenzimáticas , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Imunoeletrônica , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Nervo Óptico/irrigação sanguínea , Nervo Óptico/citologia , Nervo Óptico/virologia , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
We investigated the long-term effects of physiological levels of leptin produced by gene therapy on body weight (BW) and expression of genes that encode orexigenic and anorexigenic peptides in the hypothalamus. Recombinant adeno-associated viral vector (rAAV), a non-pathogenic and non-immunogenic vector, encoding leptin (betaOb) was generated and administered iv to ob/ob mice lacking endogenous leptin. Whereas the lowest dose of rAAV-betaOb (6x10(9) particles) was ineffective, the middle dose (6x10(10) particles) curbed BW gain without affecting food consumption for 75 days of observation. A ten-fold higher dose (6x10(11) particles) resulted in increased blood leptin levels and suppressed both BW gain and food consumption throughout the duration of the experiment. rAAV-betaOb doses that either curbed BW without affecting food consumption or evoked BW loss and reduced food intake, decreased the expression of genes encoding the orexigenic peptides, neuropeptide Y and agouti-related peptide in the ARC, and the two doses were equally effective. Concomitantly, the expression of genes encoding the anorexigenic peptide, alpha-melanocyte stimulating hormone and cocaine-and-amphetamine regulatory transcript, was augmented with the latter gene displaying a dose-dependant response. These results document the efficacy of delivering biologically active leptin for extended periods by an iv injection of rAAV-betaOb and show that physiological leptin concentrations simultaneously exert a tonic inhibitory effect on orexigenic and a stimulatory effect on anorexigenic signaling in the hypothalamus. This intricate dynamic interplay induced by leptin regulates BW with or without an effect on food intake in leptin-deficient ob/ob mice. Further, these results suggest that gene therapy is an effective mode of delivery to the hypothalamus of those therapeutic proteins that cross the blood-brain barrier to ameliorate neuroendocrine disorders.
Assuntos
Leptina/metabolismo , Proteínas do Tecido Nervoso/genética , Neuropeptídeo Y/genética , Pró-Opiomelanocortina/genética , Proteínas/genética , alfa-MSH/genética , Proteína Relacionada com Agouti , Animais , Peso Corporal , Dependovirus , Regulação da Expressão Gênica , Vetores Genéticos , Peptídeos e Proteínas de Sinalização Intercelular , Leptina/sangue , Leptina/genética , Masculino , Camundongos , Camundongos Obesos , Fatores de TempoRESUMO
The weight-reducing effects of leptin are predominantly mediated through the hypothalamus in the brain. Gene therapy strategies designed for weight control have so far tested the short-term effect of peripherally delivered viral vectors encoding the leptin gene. In order to circumvent the multiple peripheral effects of hyperleptinemia and to overcome the age-related development of leptin resistance due to multiple factors, including defective leptin transport across the blood brain barrier, we determined whether delivery of viral vectors directly into the brain is a viable therapeutic strategy for long-term weight control in normal wild-type rats. A recombinant adeno-associated virus (rAAV) vector encoding rat leptin (Ob) cDNA was generated (rAAV-betaOb). When administered once intracerebroventricularly (i.c.v.), rAAV-betaOb suppressed the normal time-related weight gain for extended periods of time in adult Sprague-Dawley rats. The vector expression was confirmed by immunocytochemical localization of GFP and RT-PCR analysis of leptin in the hypothalamus. This sustained restraint on weight gain was not due to shifts in caloric consumption because food-intake was similar in rAAV-betaOb-treated and rAAV-GFP-treated control rats throughout the experiment. Weight gain suppression, first apparent after 2 weeks, was a result of reduced white fat depots and was accompanied by drastically reduced serum leptin and insulin concentrations in conjunction with normoglycemia. Additionally, there was a marked increase in uncoupling protein-1 (UCP1) mRNA expression in brown adipose tissue, thereby indicating increased energy expenditure through thermogenesis. Seemingly, a selective enhancement in energy expenditure following central delivery of the leptin gene is a viable therapeutic strategy to control the age-related weight gain and provide protection from the accompanying multiple peripheral effects of hyperleptinemia and hyperinsulinemia.
Assuntos
Tecido Adiposo/metabolismo , Comportamento Alimentar , Antagonistas da Insulina/administração & dosagem , Insulina/sangue , Leptina/genética , Obesidade/prevenção & controle , Aumento de Peso/genética , Adenoviridae/genética , Tecido Adiposo/fisiopatologia , Tecido Adiposo Marrom/metabolismo , Envelhecimento/sangue , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Composição Corporal/genética , Proteínas de Transporte/metabolismo , Feminino , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas de Fluorescência Verde , Hipotálamo/metabolismo , Imuno-Histoquímica , Injeções Intraventriculares , Canais Iônicos , Leptina/administração & dosagem , Leptina/biossíntese , Leptina/sangue , Proteínas Luminescentes/análise , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais , Obesidade/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Desacopladora 1RESUMO
Brain-derived neurotrophic factor (BDNF) was expressed via injection of viral vector into the substantia nigra pars compacta (SNc) to investigate its influence on nigrostriatal dopaminergic activity and locomotor behavior. The recombinant adeno-associated virus (rAAV) vector, pTR-BDNFmyc, incorporated the neuron-specific enolase (NSE) promoter and the internal ribosome entry site (IRES) element driving expression of both epitope-tagged BDNF and green fluorescent protein (GFP) bicistronically. The control vector, pTR-UF4, incorporated NSE promoter-driven GFP expression only. Transgene expression persisted in both vector groups throughout the 9 month course of the study. Partial 6-hydroxydopamine (6-OHDA) lesions were conducted in the SNc ipsilateral to, and 6 months after, transduction with either the pTR-BDNFmyc or the pTR-UF4. Transgenic BDNFmyc had no effect on the number of tyrosine hydroxylase (TH)-labeled neurons in the SNc after 6-OHDA-lesions, but did block the amphetamine-induced, ipsiversive, turning-behavior caused by the lesion in the pTR-UF4 group. The BDNFmyc-transduced group also demonstrated more locomotor activity and rotational activity contralateral to the lesioned side than did the pTR-UF4-transduced group. Long-term, stable expression of BDNF can therefore modulate locomotor activity without significantly affecting nigrostriatal dopaminergic survival.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Técnicas de Transferência de Genes , Atividade Motora/genética , Substância Negra/metabolismo , Adrenérgicos , Anfetamina/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Vetores Genéticos/genética , Humanos , Oxidopamina , Doença de Parkinson/terapia , Ratos , Ratos Sprague-Dawley , Substância Negra/efeitos dos fármacosRESUMO
Administration of nerve growth factor (NGF) by intracerebroventricular infusion or transplantation of NGF-secreting cells to the basal forebrain improves spatial memory in aged animals. Using the adeno-associated virus (AAV) vector system, basal forebrain neurons were transduced to produce NGF ectopically for long intervals (at least 9 months). Rats received intraseptal injections of either the control vector, pTR-UF4, or the pTR-NGFmyc at 3 months of age, prior to testing their performance in the Morris water task. An age-related decrease in the acquisition of the hidden platform location was found at 12 months of age in the pTR-UF4 control group, but not in the pTR-NGFmyc group. Further, when compared to 3 month old untreated animals, the control group, but not the pTR-NGFmyc group, was impaired at 12 months of age. Concomitant to preventing age-related memory deficits, the NGF gene transfer increased cholinergic neuron size by 34% in the medial septum. This approach may therefore represent a viable therapy for age-related dementia involving dysfunction in cholinergic activity and memory, such as Alzheimer's disease.
Assuntos
Envelhecimento/psicologia , Transtornos da Memória/prevenção & controle , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Prosencéfalo/efeitos dos fármacos , Percepção Espacial/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Tamanho Celular , Colina O-Acetiltransferase/metabolismo , Expressão Gênica , Técnicas de Transferência de Genes , Masculino , Memória/fisiologia , Fator de Crescimento Neural/genética , Neurônios/citologia , Neurônios/enzimologia , Prosencéfalo/citologia , Ratos , Ratos Sprague-Dawley , Transgenes/fisiologiaRESUMO
The expression of GAP-43 was modulated genetically in the adult rat nigrostriatal or septohippocampal pathway using recombinant adeno-associated virus (rAAV) vectors incorporating the neuron specific enolase (NSE) promoter and either a rat GAP-43 cDNA or the corresponding antisense sequence. Bicistronic expression of green fluorescent protein (GFP) enabled us to evaluate transduced neurons selectively. Single injections of rAAV into the substantia nigra pars compacta (SNc) transduced both dopaminergic and non-dopaminergic neurons stably for the 3-month duration of the study. Transduction with the GAP-43 vector in this region: (1) increased GAP-43 mRNA levels 2-fold compared to controls; (2) led to GAP-43 immunoreactivity in neuronal perikarya, axons, and dendrites that was not observed otherwise; and (3) resulted in GAP-43/ GFP-positive axons that were traced to the striatum where they formed clusters of aberrant nets. The GAP-43 antisense vector, in contrast, decreased neuropil GAP-43 immunoreactivity compared to controls in the SNc. In septum, injections of the GAP-43 expressing vector also caused aberrant clusters of GAP-43 labelled fibers in terminal fields, i.e., fornix and hippocampus, that were not observed in control tissues. It therefore appears that rAAV vectors provide a novel approach for modulating intraneuronal GAP-43 expression in the adult brain.
Assuntos
Encéfalo/fisiologia , Proteína GAP-43/biossíntese , Técnicas de Transferência de Genes , Degeneração Neural , Regeneração Nervosa , Neurônios/fisiologia , Vias Aferentes/fisiologia , Animais , Encéfalo/metabolismo , Corpo Estriado/fisiologia , Proteínas de Fluorescência Verde , Hipocampo/fisiologia , Indicadores e Reagentes , Proteínas Luminescentes/análise , Ratos , Ratos Sprague-Dawley , Septo Pelúcido/fisiologia , Substância Negra/fisiologiaRESUMO
This study examined the 'humanized, red-shifted' version of the jellyfish Aequorea victoria green fluorescent protein (hrGFP) as a novel reporter for in vivo gene transfer studies in the cochlea using adeno-associated virus (AAV) vectors. Approximately 10(5) AAV vectors containing the hrGFP reporter gene were infused over 2 days or 1 week into the cochlea of the guinea pig via an osmotic minipump. Saline infused, non-infused, as well as AAV-beta-galactosidase infused guinea pigs served as the negative controls. The hrGFP transgene expression was detected as moderate intensity fluorescence easily distinguished from the background. Increased fluorescence was seen in the spiral ganglion, spiral ligament, spiral limbus, organ of Corti, and Reissner's membrane of the AAV-hrGFP infused animals. Control animals showed minimal fluorescence throughout the cochlea. Comparison of the 2 day and 1 week AAV-hrGFP infused animals showed qualitatively increased fluorescence in the 2 day animals. Background autofluorescence in the stria vascularis was noted in both the experimental and the control animals. In addition, fluorescence was detected in the contralateral cochlea of the AAV-hrGFP infused animals. Subsequent PCR analysis confirmed the presence of viral particles in the AAV-hrGFP infused cochlea as well as in the brain and the contralateral cochlea. This finding has important implications for the eventual implementation of cochlear gene therapy. The results not only reinforce the need to assess the introduction and expression of foreign genes in the target cochlea but also consider issues of viral spread, safety, and modes of gene delivery. This study establishes hrGFP as an effective reporter of gene transfer and transgene expression in the cochlea. GFP's small gene size, stability, ease of detection, and potential for diverse biological applications will be invaluable for a variety of future gene transfer and expression studies in the cochlea.
Assuntos
Cóclea/metabolismo , Dependovirus/genética , Regulação Viral da Expressão Gênica/genética , Técnicas de Transferência de Genes , Genes Reporter/genética , Vetores Genéticos/genética , Proteínas Luminescentes/genética , Animais , Cóclea/citologia , Primers do DNA/análise , Dependovirus/enzimologia , Terapia Genética , Proteínas de Fluorescência Verde , Cobaias , Masculino , Microscopia de Fluorescência , Órgão Espiral/metabolismo , Reação em Cadeia da Polimerase , Cifozoários , Gânglio Espiral da Cóclea/metabolismoRESUMO
BACKGROUND: We have used wild-type and recombinant adeno-associated virus-2 (AAV) to study transduction, replication efficiencies, functional protein expression, and gene delivery to vascular cells in vitro and in vivo. METHODS: Recombinant adeno-associated virus-2 (rAAV) plasmids (ranging in size to 110% of wild-type AAV) driven by 6 distinct promoters upstream of a beta-galactosidase cassette were effectively used for generation of replication-deficient virus, with titers consistently ranging from 2.5 x 10(5) IU/mL. AAV infectivity and replication in human umbilical vein endothelial cells (HUVEC) were unrelated to cellular proliferative index establishing the potential utility of the virus for transduction of quiescent vascular cells. Long-term cultures of AAV-infected HUVEC established the presence of episomal forms at 18 days, although chromosome 19-specific integration was not evident. Functional beta-galactosidase activity approximately 400% above control was evident in HUVEC using either a murine collagen alpha 1(I) promoter (pTRCol alpha 1(I) beta) or CMV promoter (pTRCMV beta). RESULTS: Based on these initial data, in vivo studies were completed using a rat carotid artery model. Both wild-type AAV (titers -1X10(9) IU/mL) and rAAV (pTRCol alpha 1(I) beta or pTRCMV beta) efficiently infected vascular cells in vivo with endothelial and vascular smooth muscle cell transduction frequencies approaching 90% as judged by DNA in situ polymerase chain reaction, with no evidence for disrupted vessel architecture. Protein expression using total vessel extracts at 48 hours postinfection demonstrated 20-fold increase in functional beta-galactosidase activity using pTRCol alpha 1(I) beta compared to saline-injected controls vessels (799 +/- 236 microU/mg protein vs 40.7 +/- 17 microU/mg protein). CONCLUSIONS: These data provide the first evidence that rAAV may be adapted for directed high-level transgene delivery and expression into normally quiescent vascular endothelial and smooth muscle cells both in vitro and in vivo.