RESUMEN
For ethical and safety reasons it is critical to develop easily implemented assays with high sensitivity and specificity for gene doping surveillance. Two nested quantitative real-time PCR (qPCR) assays were developed that target the human EPO (hEPO) cDNA sequence in a circular form, representative of recombinant adeno-associated viral (rAAV) vector genomes found in vivo. Through an interlaboratory evaluation, the assays were validated and utilized in an in vitro blinded study. These assays are specific and extremely sensitive with a limit of detection (LOD) of 1 copy of circular plasmid DNA and a limit of quantification (LOQ) of 10 to 20 copies in the presence of 500 ng of human genomic DNA (hgDNA) extracted from WBCs. Additionally, using the two nested qPCR assays in a non-human primate study, where macaques were injected intramuscularly with a rAAV8 vector harboring a promoterless hEPO cDNA sequence, the viral vector was detected 8 to 14 weeks post-injection in macaque WBCs. The high sensitivity of the nested qPCR approach along with the capability of quantifying target DNA, make this approach a reliable tool for gene doping surveillance and the monitoring of exogenous DNA sequences.
Asunto(s)
Eritropoyetina/genética , Reacción en Cadena de la Polimerasa/métodos , Transgenes , Animales , ADN/sangre , ADN/genética , ADN/aislamiento & purificación , Doping en los Deportes/prevención & control , Eritropoyetina/análisis , Eritropoyetina/sangre , Vectores Genéticos , Humanos , Macaca , Macaca fascicularis , Masculino , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Haemophilia B, or factor IX deficiency, is a X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5-30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral and adenoviral vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.
Asunto(s)
Dependovirus/genética , Factor IX/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Hemofilia B/terapia , Hígado/metabolismo , Alanina Transaminasa/sangre , Alanina Transaminasa/metabolismo , Animales , División Celular , ADN sin Sentido/genética , ADN sin Sentido/metabolismo , Factor IX/metabolismo , Expresión Génica , Terapia Genética , Hemofilia B/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Legitimate uses of gene transfer technology can benefit from sensitive detection methods to determine vector biodistribution in pre-clinical studies and in human clinical trials, and similar methods can detect illegitimate gene transfer to provide sports-governing bodies with the ability to maintain fairness. Real-time PCR assays were developed to detect a performance-enhancing transgene (erythropoietin, EPO) and backbone sequences in the presence of endogenous cellular sequences. In addition to developing real-time PCR assays, the steps involved in DNA extraction, storage and transport were investigated. By real-time PCR, the vector transgene is distinguishable from the genomic DNA sequence because of the absence of introns, and the vector backbone can be identified by heterologous gene expression control elements. After performance of the assays was optimized, cynomolgus macaques received a single dose by intramuscular (IM) injection of plasmid DNA, a recombinant adeno-associated viral vector serotype 1 (rAAV1) or a rAAV8 vector expressing cynomolgus macaque EPO. Macaques received a high plasmid dose intended to achieve a significant, but not life-threatening, increase in hematocrit. rAAV vectors were used at low doses to achieve a small increase in hematocrit and to determine the limit of sensitivity for detecting rAAV sequences by single-step PCR. DNA extracted from white blood cells (WBCs) was tested to determine whether WBCs can be collaterally transfected by plasmid or transduced by rAAV vectors in this context, and can be used as a surrogate marker for gene doping. We demonstrate that IM injection of a conventional plasmid and rAAV vectors results in the presence of DNA that can be detected at high levels in blood before rapid elimination, and that rAAV genomes can persist for several months in WBCs.
Asunto(s)
ADN Viral/sangre , Dependovirus/genética , Doping en los Deportes , Eritropoyetina/sangre , Eritropoyetina/genética , Vectores Genéticos , Farmacocinética , Plásmidos/sangre , Transgenes , Animales , Sistemas de Computación , ADN/sangre , Técnicas de Transferencia de Gen , Inyecciones Intramusculares , Leucocitos/química , Macaca fascicularis , Reacción en Cadena de la Polimerasa , Factores de TiempoRESUMEN
Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.
Asunto(s)
Dependovirus , Factor IX/genética , Terapia Genética , Vectores Genéticos , Hemofilia B/terapia , Animales , Anticuerpos/sangre , Tiempo de Sangría , Transformación Celular Viral , Modelos Animales de Enfermedad , Perros , Humanos , Hígado , Ratones , Ratones Endogámicos C57BL , Recombinación GenéticaRESUMEN
Dopamine-deficient mice (DA-/- ), lacking tyrosine hydroxylase (TH) in dopaminergic neurons, become hypoactive and aphagic and die by 4 weeks of age. They are rescued by daily treatment with L-3,4-dihydroxyphenylalanine (L-DOPA); each dose restores dopamine (DA) and feeding for less than 24 hr. Recombinant adeno-associated viruses expressing human TH or GTP cyclohydrolase 1 (GTPCH1) were injected into the striatum of DA-/- mice. Bilateral coinjection of both viruses restored feeding behavior for several months. However, locomotor activity and coordination were partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior.
Asunto(s)
Adenoviridae/genética , Dopamina/deficiencia , Conducta Alimentaria/fisiología , Técnicas de Transferencia de Gen , Vectores Genéticos , Animales , Catecolaminas/metabolismo , GTP Ciclohidrolasa/genética , Humanos , Inmunohistoquímica , Isoenzimas/genética , Levodopa/farmacología , Enfermedades Metabólicas/mortalidad , Enfermedades Metabólicas/fisiopatología , Ratones , Ratones Endogámicos , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Recombinación Genética , Conducta Estereotipada/fisiología , Distribución Tisular , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Unión Competitiva , Replicación del ADN , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/metabolismo , Polirribonucleótidos/metabolismo , Proteína de Replicación A , Virus 40 de los Simios/metabolismoRESUMEN
Control of gene expression is important to gene therapy for purposes of both dosing and safety. In vivo regulation of gene expression was demonstrated following co-injection of two separate recombinant adeno-associated virus vectors, one encoding an inducible murine erythropoietin transgene and the other a transcriptional activator, directly into the skeletal muscle of adult immunocompetent mice. Transcription was controlled by systemic administration or withdrawal of tetracycline over an 18 week period, demonstrating that the two vectors were capable of transducing the same cell. Cellular or humoral immune responses against the transactivator protein were not detected.
Asunto(s)
Dependovirus/genética , Regulación de la Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/genética , Células 3T3 , Animales , Formación de Anticuerpos , Células Cultivadas , Eritropoyetina/biosíntesis , Eritropoyetina/genética , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Hematócrito , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/metabolismo , Regiones Promotoras Genéticas , Linfocitos T Citotóxicos/inmunología , Tetraciclina/farmacología , Transactivadores/genética , TransgenesRESUMEN
Recombinant adeno-associated virus (rAAV) vectors were evaluated for gene transfer into the skeletal muscle of adult immunocompetent mice. A study using a vector encoding nuclear localized beta-galactosidase (rAAV-nls-lacZ) examined: (i) the efficiency and duration of transgene expression; (ii) the status of the AAV genome in the transduced fibers; and (iii) the possibility of improving gene transfer by inducing muscle regeneration. In the absence of regeneration, the injection of 1.7 x 10(7) particles in the quadriceps resulted in gene transfer to 10-70% of myofibers. Histological analysis indicated that the vector was able to reach myofiber nuclei distant from the injection point. Cellular infiltrates were absent at early time points but became conspicuous in the vicinity of some positive fibers at 4-8 weeks and subsided by 26 weeks. Southern analysis indicated that one to three copies of the vector genome were present per cell genome equivalent. They were associated with high-molecular-weight DNA in the form of tandem oligomers or interlocked circles. Gene transfer was not facilitated in the regenerating muscle. Rather, an early inflammatory response resulted in the elimination of most positive fibers after 8 weeks. The presence of regenerated fibers with beta-galactosidase-positive nuclei suggested that myoblasts had been transduced and were able to fuse to form new fibers. Gene transfer in the absence of immune reactions against the transgene product was studied by injecting mice with a rAAV carrying the murine erythropoietin (mEpo) cDNA. Dose-dependent elevation in the hematocrit was measured for over 200 days and corresponded to 5- to 20-fold increases in plasma Epo levels. We conclude that AAV vectors efficiently and stably transduce post-mitotic muscle fibers and myoblasts in vivo.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Músculo Esquelético/metabolismo , Transfección/genética , Animales , Southern Blotting , Dependovirus/metabolismo , Eritropoyetina/genética , Eritropoyetina/metabolismo , Femenino , Regulación Viral de la Expresión Génica/genética , Genoma Viral , Hematócrito , Histocitoquímica , Operón Lac/genética , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/citología , Miofibrillas/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
As a potential treatment for Parkinson's disease, viral vector-mediated over-expression of striatal L-aromatic amino acid decarboxylase was tested in an attempt to facilitate the production of therapeutic levels of dopamine after peripheral L-dihydroxyphenylalanine administration. The results of microdialysis and enzyme activity assays indicate that striatal decarboxylation of peripherally administered L-dihydroxyphenylalanine was enhanced by recombinant adeno-associated virus-mediated gene transfer of L-aromatic amino acid decarboxylase in unilateral 6-hydroxydopamine-lesioned rats. This gene transfer-induced increase in striatal decarboxylase activity was shown to remain undiminished over a six-month period and transgene expression was demonstrated to persist for at least one year. Unlike previous approaches involving delivery of either tyrosine hydroxylase, or tyrosine hydroxylase and L-aromatic amino acid decarboxylase transgenes together to accomplish unregulated dopamine delivery, the current study proposes a pro-drug strategy (peripheral L-dihydroxyphenylalanine administration after L-aromatic amino acid decarboxylase transduction). This strategy for dosage control could potentially allow lowered L-dihydroxyphenylalanine doses and potentially obviate complicated transcriptional regulation paradigms. These data suggest that the use of the non-pathogenic adeno-associated virus to transfer the L-aromatic amino acid decarboxylase gene into the striatum of Parkinson's disease patients may be an attractive gene therapy strategy.
Asunto(s)
Adenoviridae/genética , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Cuerpo Estriado/enzimología , Técnicas de Transferencia de Gen , Enfermedad de Parkinson/enzimología , Animales , Dopamina/biosíntesis , Expresión Génica/fisiología , Vectores Genéticos , Células HeLa , Humanos , Masculino , Ratas , Ratas Endogámicas F344 , Recombinación Genética , Factores de Tiempo , Transducción Genética/fisiologíaRESUMEN
Several gene delivery vehicles are being developed for somatic gene therapy and each of these vectors has unique properties which makes them appropriate for different human disease applications. Recombinant adeno-associated viral (rAAV) vectors are proving themselves to be safe and efficacious for the long-term expression of proteins and correction of genetic diseases following a single administration. The increasing number of tissues and diseases being targeted with rAAV vectors demonstrates their versatility and has resulted in different approaches for enhancing vector performance. Improving the methods for large-scale manufacturing, and accumulating safety and efficacy data in animals and humans are areas of intense research.
Asunto(s)
Dependovirus/genética , Terapia Genética , Vectores Genéticos , Animales , Biotecnología , Técnicas de Transferencia de Gen , Ingeniería Genética , Humanos , Recombinación GenéticaRESUMEN
Regulated DNA deletions are known to occur to thousands of specific DNA segments in Tetrahymena during macronuclear development. In this study we determined the precision of this event by examining the junction sequences produced by three different deletions in many independent caryonidal lines. 0.9 kb deletions in region M produce at least 3 types of junction sequences, of which two have been determined and found to be different by 4 bp. The alternative 0.6 kb deletions in this region are much less variable. 1.1 kb deletions in region R, known from a previous study to be slightly variable, produce two types of junction sequences which are different from each other by 3 bp. Thus, developmentally regulated deletions in Tetrahymena can produce sequence microheterogeneity at their junctions. This process contributes significantly to the diversification of Tetrahymena's somatic genome.
Asunto(s)
Deleción Cromosómica , Tetrahymena/genética , Animales , Secuencia de Bases , Southern Blotting , ADN/genética , Variación Genética , Datos de Secuencia Molecular , Sondas de OligonucleótidosRESUMEN
We have demonstrated that when the covalently joined ends of linear adeno-associated virus (AAV) DNA are resolved in vitro, the virus-encoded Rep protein becomes covalently attached to the 5' ends of the DNA. The covalent bond is between a tyrosine residue of the AAV Rep protein and a 5' phosphate of a thymidine residue in the AAV genome. Only the Rep protein encoded by the AAV p5 promoter, Rep68, was capable of becoming covalently attached to the ends of the AAV genome; the Rep proteins encoded by the p19 promoter were not. We also investigated some of the requirements for the complete in vitro resolution reaction. Inhibitor studies suggested that terminal resolution required DNA polymerase delta, ATP, and the deoxyribonucleoside triphosphates but did not require the remaining ribonucleoside triphosphates, DNA polymerase alpha, RNA polymerase II, or topoisomerases I and II. Finally, purified AAV Rep68, when added to the crude cytosol from uninfected HeLa cells, was sufficient for resolution. This suggested that terminal resolution relies on host enzymes and the virus-encoded p5 Rep proteins.
Asunto(s)
Replicación del ADN , Dependovirus/genética , Genes Virales , Proteínas Virales/metabolismo , Sitios de Unión , ADN Polimerasa III , ADN Viral/genética , ADN Viral/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Células HeLa/metabolismo , Humanos , Modelos Genéticos , Hibridación de Ácido Nucleico , Plásmidos , Unión Proteica , TirosinaRESUMEN
We have developed an assay for a key step in the replication of adeno-associated virus (AAV) DNA. We demonstrate the covalently joined ends of linear AAV DNA can be resolved in vitro to the open duplex configuration. Only extracts prepared from human cells that have been infected with both adenovirus and AAV are capable of carrying out the reaction. The reaction is initiated by a site-specific and strand-specific endonucleolytic cut at a terminal resolution site near the end of the AAV terminal palindrome. During resolution the orientation of the terminal palindrome is inverted, and the 3' viral strand is extended by DNA synthesis. The size of the newly synthesized 3' strand is nearly identical to that found in viral particles. These observations provide direct biochemical evidence for an essential step in the model for AAV DNA replication.
Asunto(s)
Replicación del ADN , ADN Viral/genética , Dependovirus/genética , Secuencia de Bases , Sistema Libre de Células , Células HeLa , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Plásmidos , Mapeo Restrictivo , Virión/genéticaRESUMEN
Previous work has demonstrated that viral vector mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF), when administered prior to a striatal injection of the specific neurotoxin, 6-hydroxydopamine (6-OHDA), can protect nigral dopamine (DA) neurons from cell death. When considering gene therapy for Parkinson's disease (PD), vector delivery prior to the onset of neuropathology is not possible and chronic delivery will likely be necessary in a GDNF-based PD therapy. The present study was undertaken to determine if GDNF delivered via a recombinant adeno-associated viral vector (rAAV) could affect nigral DA cell survival when initiated just after the administration of striatal 6-OHDA. The onset of rAAV-mediated GDNF transgene expression near the substantia nigra was determined to begin somewhere between 1 and 7 days after the 6-OHDA injection and subsequent vector administration. The cell survival data indicate that rAAV-GDNF delivery results in a highly significant sparing of nigral DA neurons. These data indicate that a single delivery of rAAV encoding GDNF is efficacious when delivered after the onset of progressive degeneration in a rat model of PD.
Asunto(s)
Dependovirus/genética , Dopamina/análisis , Terapia Genética , Vectores Genéticos/uso terapéutico , Degeneración Nerviosa , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Enfermedad de Parkinson Secundaria/terapia , Sustancia Negra/patología , Animales , Citomegalovirus/genética , Genes Reporteros , Vectores Genéticos/genética , Factor Neurotrófico Derivado de la Línea Celular Glial , Operón Lac , Masculino , Proteínas del Tejido Nervioso/biosíntesis , Oxidopamina/toxicidad , Enfermedad de Parkinson Secundaria/patología , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Simpaticolíticos/toxicidadRESUMEN
Viral vectors have recently been used successfully to transfer genes and express different proteins in the brain. This review discusses the requirements to consider human clinical trials in which recombinant adeno-associated virus vectors are used to transfer the genes necessary to produce l-dihydroxyphenylalanine (l-dopa) directly into the striatum of Parkinson's patients. Preclinical data that apply to the criteria defined as prerequisite for clinical trials are discussed. Thus, in animal models using recombinant adeno-associated virus vectors it has been demonstrated that l-dopa can be synthesized in the striatum after in vivo transduction. In addition, these l-dopa levels are sufficient to affect behavior in a dopamine-deficient animal model, the expression is extremely long-lasting, and the ability to transcriptionally regulate tyrosine hydroxylase has been demonstrated but not fully characterized. However, while immune responses to recombinant adeno-associated virus infection in the periphery have been studied, direct assessment of the potential immune response in the brain has not been sufficiently defined. Therefore, the rationale for delivering l-dopa directly to the striatum to treat Parkinson's disease is sound and the preclinical data are promising but all the issues surrounding this strategy are not resolved.
Asunto(s)
Adenoviridae , Antiparkinsonianos/metabolismo , Cuerpo Estriado , Terapia Genética , Vectores Genéticos , Levodopa/genética , Animales , Humanos , Proteínas Recombinantes/genéticaRESUMEN
A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson's disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson's disease.
Asunto(s)
Terapia Genética/métodos , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/genética , Enfermedad de Parkinson Secundaria/terapia , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Expresión Génica , Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial , Células HeLa , Humanos , Degeneración Nerviosa , Proteínas del Tejido Nervioso/metabolismo , Oxidopamina/toxicidad , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/patología , Ratas , Ratas Endogámicas F344 , Recombinación Genética , Sustancia Negra/patologíaRESUMEN
The skeletal muscle provides a very permissive physiological environment for adeno-associated virus (AAV) type 2-mediated gene transfer. We have studied the early steps leading to the establishment of permanent transgene expression, after injection of recombinant AAV (rAAV) particles in the quadriceps muscle of mice. The animals received an rAAV encoding a secreted protein, murine erythropoietin (mEpo), under the control of the human cytomegalovirus major immediate-early promoter and were sacrificed between 1 and 60 days after injection. The measurement of plasma Epo levels and of hematocrits indicated a progressive increase of transgene expression over the first 2 weeks, followed by a stabilization at maximal plateau values. The rAAV sequences were analyzed by Southern blotting following neutral or alkaline gel electrophoresis of total DNA from injected muscles. While a high number of rAAV sequences were detected during the first 5 days following the injection, only a few percent of these sequences was retained in the animals analyzed after 2 weeks, in which transgene expression was maximal. Double-stranded DNA molecules resulting from de novo second-strand synthesis were detected as early as day 1, indicating that this crucial step of AAV-mediated gene transfer is readily accomplished in the muscle. The templates driving stable gene expression at later time points are low in copy number and structured as high-molecular-weight concatemers or interlocked circles. The presence of the circular form of the rAAV genomes at early time points suggests that the molecular transformations involved in the formation of stable concatemers may involve a rolling-circle type of DNA replication.
Asunto(s)
ADN Viral/química , Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Músculo Esquelético/metabolismo , Animales , Replicación del ADN , Ratones , Ratones Endogámicos BALB CRESUMEN
To achieve local, continuous L-DOPA delivery in the striatum by gene replacement as a model for a gene therapy for Parkinson's disease, the present studies used high titer purified recombinant adeno-associated virus (rAAV) containing cDNAs encoding human tyrosine hydroxylase (hTH) or human GTP-cyclohydrolase I [GTPCHI, the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis] or both to infect the 6-OHDA denervated rat striatum. Striatal TH and GTPCHI staining was observed 3 weeks after rAAV transduction, with little detectable perturbation of the tissue. Six months after intrastriatal rAAV transduction, TH staining was present but apparently reduced compared with the 3 week survival time. In a separate group of animals, striatal TH staining was demonstrated 1 year after rAAV transduction. Double staining studies using the neuronal marker NeuN indicated that >90% of rAAV-transduced cells expressing hTH were neurons. Microdialysis experiments indicated that only those lesioned animals that received the mixture of MD-TH and MD-GTPCHI vector displayed BH4 independent in vivo L-DOPA production (mean approximately 4-7 ng/ml). Rats that received the hTH rAAV vector alone produced measurable L-DOPA (mean approximately 1-4 ng/ml) only after receiving exogenous BH4. L-Aromatic amino acid decarboxylase blockade, but not 100 mM KCl-induced depolarization, enhanced L-DOPA overflow, and animals in the non-hTH groups (GTPCHI and alkaline phosphatase) yielded minimal L-DOPA. Although elevated L-DOPA was observed in animals that received mixed hTH and hGTPCHI rAAV vectors, there was no reduction of apomorphine-induced rotational behavior 3 weeks after intrastriatal vector injection. These data demonstrate that purified rAAV, a safe and nonpathogenic viral vector, mediates long-term striatal hTH transgene expression in neurons and can be used to successfully deliver L-DOPA to the striatum.
Asunto(s)
Dependovirus , GTP Ciclohidrolasa/genética , Técnicas de Transferencia de Gen , Enfermedad de Parkinson Secundaria/terapia , Tirosina 3-Monooxigenasa/genética , Animales , Apomorfina , Conducta Animal/efectos de los fármacos , Cuerpo Estriado/citología , Cuerpo Estriado/enzimología , Modelos Animales de Enfermedad , Dopaminérgicos/metabolismo , Agonistas de Dopamina , Regulación Enzimológica de la Expresión Génica , Humanos , Levodopa/genética , Levodopa/metabolismo , Masculino , Microdiálisis , Neuronas/enzimología , Enfermedad de Parkinson Secundaria/enzimología , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes/genética , RotaciónRESUMEN
We previously demonstrated that the adeno-associated virus (AAV) Rep68 and Rep78 proteins are able to nick the AAV origin of DNA replication at the terminal resolution site (trs) in an ATP-dependent manner. Using four types of modified or mutant substrates, we now have investigated the substrate requirements of Rep68 in the trs endonuclease reaction. In the first kind of substrate, portions of the hairpinned AAV terminal repeat were deleted. Only deletions that retained virtually all of the small internal palindromes of the AAV terminal repeat were active in the endonuclease reaction. This result confirmed previous genetic and biochemical evidence that the secondary structure of the terminal repeat was an important feature for substrate recognition. In the second type of substrate, the trs was moved eight bases further away from the end of the genome. The mutant was nicked at a 50-fold-lower frequency relative to a wild-type origin, and the nick occurred at the correct trs sequence despite its new position. This finding indicated that the endonuclease reaction required a specific sequence at the trs in addition to the correct secondary structure. It also suggested that the minimum trs recognition sequence extended three bases from the cut site in the 3' direction. The third type of substrate harbored mismatched base pairs at the trs. The mismatch substrates contained a wild-type sequence on the strand normally cut but an incorrect sequence on the complementary strand. All of the mismatch mutants were capable of being nicked in the presence of ATP. However, there was substantial variation in the level of activity, suggesting that the sequence on the opposite strand may also be recognized during nicking. Analysis of the mismatch mutants also suggested that a single-stranded trs was a viable substrate for the enzyme. This interpretation was confirmed by analysis of the fourth type of substrate tested, which contained a single-stranded trs. This substrate was also cleaved efficiently by the enzyme provided that the correct strand was present in the substrate. In addition, the single-stranded substrate no longer required ATP as a cofactor for nicking. Finally, all of the substrates with mutant trss bound the Rep protein as efficiently as the wild-type did. This finding indicated that the sequence at the cut site was not involved in recognition of the terminal repeat for specific binding by the enzyme. We concluded that substrate recognition by the AAV Rep protein involves at least two and possibly as many as four features of the AAV terminal repeat.(ABSTRACT TRUNCATED AT 400 WORDS)