RESUMEN
The embryonic CNS readily undergoes regeneration, unlike the adult CNS, which has limited axonal repair after injury. Here we tested the hypothesis that retinoic acid receptor beta2 (RARbeta2), critical in development for neuronal growth, may enable adult neurons to grow in an inhibitory environment. Overexpression of RARbeta2 in adult rat dorsal root ganglion cultures increased intracellular levels of cyclic AMP and stimulated neurite outgrowth. Stable RARbeta2 expression in DRG neurons in vitro and in vivo enabled their axons to regenerate across the inhibitory dorsal root entry zone and project into the gray matter of the spinal cord. The regenerated neurons enhanced second-order neuronal activity in the spinal cord, and RARbeta2-treated rats showed highly significant improvement in sensorimotor tasks. These findings show that RARbeta2 induces axonal regeneration programs within injured neurons and may thus offer new therapeutic opportunities for CNS regeneration.
Asunto(s)
Regeneración Nerviosa/fisiología , Neuronas Aferentes/metabolismo , Receptores de Ácido Retinoico/metabolismo , Médula Espinal/metabolismo , Animales , Células Cultivadas , Ganglios Espinales/lesiones , Ganglios Espinales/metabolismo , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratas , Ratas Wistar , Recuperación de la Función/fisiologíaRESUMEN
Pharmacological dopamine replacement therapies provide the most well-established treatments for Parkinson's disease (PD). However, these long-term treatments can lead to motor complications and off-target effects. ProSavin(®), a lentiviral vector (LV)-based gene therapy approach aimed at restoring local and continuous dopamine production, through delivery of three enzymes in the dopamine biosynthesis pathway, was demonstrated to be safe and well-tolerated in a phase I/II clinical study of patients with advanced PD. Although improvements in motor behaviour were observed, the data indicated that higher levels of dopamine replacement might be required to maximize benefit. We attempted to increase production of dopamine, and its precursor L-Dopa in LV-transduced cells, by optimizing the gene order in the ProSavin expression cassette, and by creating fusions of two or three of the transgenes, using linker sequences. In vitro analysis showed that several gene arrangements provided significantly increased dopamine and/or L-Dopa production compared with ProSavin, and that LV titers and transgene expression were not affected by introducing gene fusions. One vector, equine infectious anemia virus (EIAV)-TCiA, was selected for further characterization and showed significant improvements in dopamine and L-Dopa production compared with ProSavin, in human neuronal cells. Further characterization of EIAV-TCiA demonstrated expression of all three dopamine enzymes in vivo and faithful delivery and integration of the expected gene expression cassette within the genome of target cells, as assessed by Northern and Southern blotting. In conclusion, we have developed a novel LV vector with an increased capacity for L-Dopa and dopamine production compared with the current ProSavin vector. Clinical evaluation of this vector will be performed to assess the benefits in patients with PD.
Asunto(s)
Dopamina/biosíntesis , Terapia Genética , Enfermedad de Parkinson/terapia , Proteínas Recombinantes de Fusión/genética , Transgenes/genética , Animales , Células Cultivadas , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Masculino , Neuronas/citología , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra. This loss leads to complete dopamine depletion in the striatum and severe motor impairment. It has been demonstrated previously that a lentiviral vector system based on equine infectious anemia virus (EIAV) gives rise to highly efficient and sustained transduction of neurons in the rat brain. Therefore, a dopamine replacement strategy using EIAV has been investigated as a treatment in the 6-hydroxydopamine (6-OHDA) animal model of PD. A self-inactivating EIAV minimal lentiviral vector that expresses tyrosine hydroxylase (TH), aromatic amino acid dopa decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1) in a single transcription unit has been generated. In cultured striatal neurons transduced with this vector, TH, AADC, and CH1 proteins can all be detected. After stereotactic delivery into the dopamine-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each enzyme and effective production of catecholamines were detected, resulting in significant reduction of apomorphine-induced motor asymmetry compared with control animals (p < 0.003). Expression of each enzyme in the striatum was observed for up to 5 months after injection. These data indicate that the delivery of three catecholaminergic synthetic enzymes by a single lentiviral vector can achieve functional improvement and thus open the potential for the use of this vector for gene therapy of late-stage PD patients.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Dopamina/biosíntesis , Vectores Genéticos/administración & dosificación , Trastornos Parkinsonianos/terapia , Animales , Descarboxilasas de Aminoácido-L-Aromático/administración & dosificación , Descarboxilasas de Aminoácido-L-Aromático/biosíntesis , Descarboxilasas de Aminoácido-L-Aromático/genética , Catecolaminas/metabolismo , Células Cultivadas , Cuerpo Estriado/patología , Cuerpo Estriado/fisiopatología , Modelos Animales de Enfermedad , GTP Ciclohidrolasa/administración & dosificación , GTP Ciclohidrolasa/biosíntesis , GTP Ciclohidrolasa/genética , Expresión Génica/efectos de los fármacos , Técnicas de Transferencia de Gen , Genes/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Riñón/citología , Riñón/metabolismo , Lentivirus/genética , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxidopamina , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/fisiopatología , Ratas , Ratas Wistar , Recuperación de la Función/efectos de los fármacos , Transgenes , Resultado del Tratamiento , Tirosina 3-Monooxigenasa/administración & dosificación , Tirosina 3-Monooxigenasa/biosíntesis , Tirosina 3-Monooxigenasa/genéticaRESUMEN
A study was conducted to investigate the effects of increasing the amount of each retroviral component on vector production. It was found that, while the components of both amphotropic and ecotropic vectors were expressed independently of each other in a transient transfection system, increasing the amount of the gag/gag-pol component resulted in a decrease in virus titres for the amphotropic particles but not ecotropic particles. Analyses of the virus stocks produced indicated that the negative effect on titres was closely linked to the availability of envelope proteins for virion incorporation. The negative effect was not observed for ecotropic particle production in 293T cells, where the ecotropic receptor was absent, but was manifested when production was conducted in 293/12 cells expressing the ecotropic receptor. This suggested that the premature interaction between envelope and receptor in producer cells could limit the amount of envelope available for virion incorporation. In designing optimal vector production systems it is essential, therefore, to balance the concentration of the vector components and to ensure that there is never an excess of Gag/Gag-Pol.
Asunto(s)
Virus de la Leucemia Murina/fisiología , Replicación Viral , Animales , Línea Celular , Productos del Gen gag/metabolismo , Productos del Gen pol/metabolismo , Cinética , Virus de la Leucemia Murina/genética , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
S2 is an accessory protein of equine infectious anaemia virus (EIAV), the function of which is unknown. In order to gain insight into the function of S2, the intracellular localization of the protein, its interaction with viral proteins and its incorporation into viral particles have been investigated. Immunolocalization of S2 revealed punctate staining in the cytoplasm and the S2 protein co-precipitated with the EIAV Gag precursor. Despite overexpression of S2 through the use of a codon-optimized sequence, there was no preferential association of S2 with EIAV particles. These data suggest that S2 may function to organize the Gag protein during particle assembly in the cytoplasm but that it is unlikely to be involved in the early stages of the virus life-cycle.
Asunto(s)
Proteínas Virales/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia MolecularRESUMEN
An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations.