RESUMO
Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is caused by mutations in tripeptidyl peptidase I (TPP-I), a pepstatin-insensitive lysosomal protease, resulting in neurodegeneration, acute seizures, visual and motor dysfunction. In vitro studies suggest that TPP-I is secreted from cells and subsequently taken up by neighboring cells, similar to other lysosomal enzymes. As such, TPP-I is an attractive candidate for enzyme replacement or gene therapy. In the present studies, we examined the feasibility of gene transfer into mouse brain using recombinant adenovirus (Ad), feline immunodeficiency virus (FIV) and adeno-associated virus (AAV) vectors expressing TPP-I, after single injections into the striatum or cerebellum. A dual TPP-I- and beta-galactosidase-expressing adenovirus vector (AdTTP-I/nlsbetagal) was used to distinguish transduced (beta-galactosidase positive) cells from cells that endocytosed secreted TTP-I. Ten days after striatal injection of AdTTP-I/nlsbetagal, beta-galactosidase-positive cells were concentrated around the injection site, corpus callosum, ependyma and choroid plexus. In cerebellar injections, beta-galactosidase expression was confined to the region of injection and in isolated neurons of the brainstem. Immunohistochemistry for TPP-I expression showed that TPP-I extended beyond areas of beta-galactosidase activity. Immunohistochemistry for TTP-I after FIVTTP-I and AAV5TTP-I injections demonstrated TPP-I in neurons of the striatum, hippocampus and Purkinje cells. For all three vectors, TPP-I activity in brain homogenates was 3-7-fold higher than endogenous levels in the injected hemispheres. Our results indicate the feasibility of vector-mediated gene transfer of TPP-I to the CNS as a potential therapy for LINCL.
Assuntos
Sistema Nervoso Central/metabolismo , Terapia Genética/métodos , Modelos Animais , Lipofuscinoses Ceroides Neuronais/terapia , Nucleotidases/genética , Adenoviridae/genética , Animais , Engenharia Genética , Vetores Genéticos/administração & dosagem , Humanos , Imuno-Histoquímica/métodos , Injeções , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Transdução Genética/métodos , Tripeptidil-Peptidase 1RESUMO
The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin.
Assuntos
Adenoviridae/metabolismo , Técnicas de Transferência de Genes , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipotálamo-Hipofisário/cirurgia , Adenoviridae/genética , Animais , Células Cultivadas , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde , Sistema Hipotálamo-Hipofisário/citologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Masculino , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Núcleo Hipotalâmico Paraventricular/virologia , Técnicas de Patch-Clamp , Neuro-Hipófise/citologia , Neuro-Hipófise/metabolismo , Neuro-Hipófise/cirurgia , Neuro-Hipófise/virologia , Ratos , Ratos Sprague-Dawley , Núcleo Supraóptico/citologia , Núcleo Supraóptico/metabolismo , Núcleo Supraóptico/virologia , TransfecçãoRESUMO
Recombinant adenoviruses are useful vectors for basic research. When the vectors are used for delineating protein function, several viruses, each containing a mutated version of the transgene are compared at the same time. However, methods to generate multiple vectors simultaneously within a short time period are cumbersome. In this report, we show that a novel backbone plasmid, when cotransfected with routinely used shuttle vectors into HEK293 cells allowed for production of recombinant viruses in an average of 14 days. The recombinant viruses had no detectable wild-type virus contamination by A549 plaque assay and only three to 300 E1a copies per 109 adenovirus genomes by a sensitive PCR-based assay. Further culturing or serial amplification did not result in wild-type revertants nor did cultures show increased levels of E1a copy number by quantitative PCR. Thus, recombinant adenovirus vectors can be produced very simply, rapidly and with little to no contaminating wild-type particles. This system should facilitate the generation of multiple genetic variants by eliminating the need for time-consuming plaque purification and the need to manipulate and screen very large plasmids. We call this the RAPAd.I system.
Assuntos
Adenoviridae/genética , Vetores Genéticos , Adenoviridae/classificação , Adenoviridae/isolamento & purificação , Linhagem Celular , Técnicas de Transferência de Genes , Genoma Viral , Humanos , Plasmídeos , TransfecçãoRESUMO
Juvenile neuronal ceroid lipofuscinosis (JNCL), Batten disease, is an autosomal recessive lysosomal storage disease associated with mutations in CLN3. CLN3 has no known homology to other proteins and a function has not yet been described. The predominant mutation in CLN3 is a 1.02 kb genomic deletion that accounts for nearly 85% of the disease alleles. In this mutation, truncation of the protein by a premature stop codon results in the classical phenotype. Additional missense and nonsense mutations have been described. Some missense substitutions result in a protracted phenotype, with delays in the onset of classical clinical features, whereas others lead to classical JNCL. In this study, we examined the effect of naturally occurring point mutations on the intracellular localization of CLN3 and their ability to complement the CLN3-deficient yeast, btn1-Delta. We also examined a putative farnesylation motif thought to be involved in CLN3 trafficking. All of the point mutations, like wild-type CLN3, were highly associated with lysosome-associated membrane protein II in non-neuronal cells and with synaptophysin in neuronal cell lines. In the yeast functional assay, point mutations correlating with a mild phenotype also demonstrated CLN3 activity, whereas the mutations associated with severe disease failed to restore CLN3 function completely. CLN3 with a mutation in the farnesylation motif trafficked normally but was functionally impaired. These data suggest that these clinically relevant point mutations, causative of Batten disease, do not affect protein trafficking but rather exert their effects by impairing protein function.
Assuntos
Ciclinas/genética , Proteínas Fúngicas/genética , Glicoproteínas de Membrana , Chaperonas Moleculares , Mutação de Sentido Incorreto , Lipofuscinoses Ceroides Neuronais/genética , Proteínas de Saccharomyces cerevisiae , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Ciclinas/metabolismo , DNA , Proteínas Fúngicas/metabolismo , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Sinaptofisina/metabolismoRESUMO
Tissue-specific ablation of gene function is possible in vivo by the Cre-loxP recombinase system. We generated transgenic mice containing a human angiotensinogen gene flanked by loxP sites (hAGT(flox)). To examine the physiologic consequences of tissue-specific loss of angiotensinogen gene function in vivo, we constructed an adenovirus expressing Cre recombinase. Studies were performed in several independent lines of hAGT(flox) mice before and after intravenous administration of either Adcre or AdbetaGal as a control. Systemic administration of Adcre caused a significant decrease in circulating human angiotensinogen and markedly blunted the pressor response to administration of purified recombinant human renin. Southern blot analysis of genomic DNA from various organs revealed that the Cre-mediated deletion was liver-specific. Further analysis revealed the absence of full-length human angiotensinogen mRNA and protein in the liver but not the kidney of Adcre mice, consistent with the liver being the target for adenoviruses administered intravenously. These studies demonstrate that extra-hepatic sources of angiotensinogen do not contribute significantly to the circulating pool of angiotensinogen and provide proof-of-principle that the Cre-loxP system can be used effectively to examine the contribution of the systemic and tissue renin-angiotensin system to normal and pathological regulation of blood pressure.
Assuntos
Angiotensinogênio/genética , Deleção de Genes , Regulação da Expressão Gênica , Integrases/genética , Proteínas Virais , Adenoviridae , Animais , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Fígado/fisiologia , Camundongos , Camundongos Transgênicos , Especificidade de ÓrgãosRESUMO
Juvenile neuronal ceroid lipofuscinosis is a lysosomal storage disease that causes visual impairment, progressive mental deterioration, and eventually death. A predominant 1.02-kb deletion as well as other mutations have been described in the CLN3 gene. Lacking significant identity with proteins of known function and no overt targeting signals within the primary amino acid sequence, accurate predictions of the intracellular location and function could not be made. Further, recent conflicting reports identified CLN3 as either a lysosomal or a mitochondrial protein. Transfection experiments using native and epitope-tagged fusion proteins were evaluated to help delineate CLN3 localization. We confirmed by immunohistochemistry and brefeldin A treatment that NH2-terminal green fluorescence protein (GFP)-CLN3 fusion proteins were retained in the Golgi apparatus, with no colocalization with mitochondrial markers. Anti-CLN3 antibodies directed against amino acids 67-90 of CLN3 were generated and shown to be specific for a 50-kDa protein in HEK 293 cells and GFP-CLN3 in transfected cells. However, cells transfected with nontagged CLN3 or carboxyl-terminal-tagged CLN3 were not immunoreactive with anti-CLN3 antibodies, suggesting that normally, the amino terminus interacts with other molecules. Thus, tags on the NH2-terminus probably inhibited these interactions and movement of CLN3 from the Golgi to more distal compartments. Also, CLN3 tagged at the COOH-terminus with either GFP or FLAG epitopes were retained in the ER, indicating a role for the COOH-terminus in trafficking. Taken together, these data confirm that CLN3 traffics through the ER and Golgi.
Assuntos
Ciclinas , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Proteínas de Saccharomyces cerevisiae , Brefeldina A/metabolismo , Células Cultivadas , Proteína Coatomer , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde , Humanos , Immunoblotting , Imuno-Histoquímica , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Chaperonas Moleculares/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Rodaminas/metabolismo , TransfecçãoRESUMO
The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis.
Assuntos
Adenoviridae , Técnicas de Transferência de Genes , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Órgão Subfornical/citologia , Núcleo Supraóptico/citologia , Animais , Western Blotting , Células Cultivadas , Canais de Potássio de Retificação Tardia , Feminino , Regulação Viral da Expressão Gênica/fisiologia , Genes Reporter , Proteínas de Fluorescência Verde , Indicadores e Reagentes/análise , Proteínas Luminescentes/análise , Masculino , Potenciais da Membrana/fisiologia , Neurônios/química , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Ratos , Ratos Sprague-Dawley , Transfecção , Replicação ViralRESUMO
Production of transgenic cattle by microinjection of DNA has been difficult and costly. To explore an alternative method, one- to four-cell bovine embryos were exposed to a replication-defective retrovirus by microinjection of retrovirus producer cells into the perivitelline space. Embryos were cultured in vitro for 3-4 days, then transferred to recipient cows for further development. Thirteen of 22 embryos recovered at 15 days gestation and each of four fetuses recovered at 90 days gestation were transgenic. Fetuses harbored between 2 and 12 proviruses, and within each fetus, identical patterns of integration were observed in seven tissues tested. Estimates of the number of proviruses per cell suggested that in three of the four fetuses, most, and possibly all, cells were transgenic. This technique should facilitate application of transgenic technology to cattle and other agriculturally important species.