RESUMEN
Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.
Asunto(s)
Dependovirus/genética , Vectores Genéticos/genética , Recombinación Genética , Retina/metabolismo , Transducción Genética , Animales , Cadherinas/genética , Cadherinas/metabolismo , Expresión Génica , Regulación Viral de la Expresión Génica , Técnicas de Transferencia de Gen , Genes Reporteros , Terapia Genética , Vectores Genéticos/administración & dosificación , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Porcinos , Transcripción Genética , TransgenesRESUMEN
Neurotrophic factors have raised hopes to be able to cure symptoms and to prevent progressive neurodegeneration in devastating neurological diseases. Gene therapy by means of viral vectors can overcome the hurdle of targeted delivery, but its current configuration is irreversible and thus much less controllable than that of classical pharmacotherapies. We thus aimed at developing a strategy allowing for both curative and controllable neurotrophic factor expression. Therefore, the short-term, intermittent and reversible expression of a neutrophic factor was evaluated for therapeutic efficacy in a slowly progressive animal model of Parkinson's disease (PD). We demonstrate that short-term induced expression of glial cell line derived neurotrophic factor (GDNF) is sufficient to provide i) substantial protection of nigral dopaminergic neurons from degeneration and ii) restoration of dopamine supply and motor behaviour in the partial striatal 6-OHDA model PD. These neurorestorative effects of GDNF lasted several weeks beyond the time of its expression. Later on, therapeutic efficacy ceased, but was restored by a second short induction of GDNF expression, demonstrating that monthly application of the inducing drug mifepristone was sufficient to maintain neuroprotective and neurorestorative GDNF levels. These findings suggest that forthcoming gene therapies for PD or other neurodegenerative disorders can be designed in a way that low frequency application of an approved drug can provide controllable and therapeutically efficient levels of GDNF or other neurotrophic factors. Neurotrophic factor expression can be withdrawn in case of off-target effects or sufficient clinical benefit, a feature that may eventually increase the acceptance of gene therapy for less advanced patients, which may profit better from such approaches.
Asunto(s)
Terapia Genética/métodos , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Actividad Motora/fisiología , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/terapia , Adenoviridae/genética , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Dopamina/metabolismo , Lateralidad Funcional/efectos de los fármacos , Lateralidad Funcional/fisiología , Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Antagonistas de Hormonas/farmacología , Mifepristona/farmacología , Actividad Motora/efectos de los fármacos , Oxidopamina/toxicidad , Enfermedad de Parkinson/etiología , Ratas , Rotación , Simpaticolíticos/toxicidad , Factores de Tiempo , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
Histone acetylation has been implicated with the pathogenesis of neuropsychiatric disorders and targeting histone deacetylases (HDACs) using HDAC inhibitors was shown to be neuroprotective and to initiate neuroregenerative processes. However, little is known about the role of individual HDAC proteins during the pathogenesis of brain diseases. HDAC1 was found to be upregulated in patients suffering from neuropsychiatric diseases. Here, we show that virus-mediated overexpression of neuronal HDAC1 in the adult mouse hippocampus specifically affects the extinction of contextual fear memories, while other cognitive abilities were unaffected. In subsequent experiments we show that under physiological conditions, hippocampal HDAC1 is required for extinction learning via a mechanism that involves H3K9 deacetylation and subsequent trimethylation of target genes. In conclusion, our data show that hippocampal HDAC1 has a specific role in memory function.
Asunto(s)
Extinción Psicológica/fisiología , Miedo/fisiología , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/fisiología , Estimulación Acústica , Animales , Benzamidas/administración & dosificación , Benzamidas/farmacología , Western Blotting , Inmunoprecipitación de Cromatina , Electrochoque , Conducta Exploratoria/fisiología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Histona Desacetilasa 1/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/administración & dosificación , Inhibidores de Histona Desacetilasas/farmacología , Inmunohistoquímica , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Microinyecciones , Neuronas/metabolismo , Equilibrio Postural/fisiología , Piridinas/administración & dosificación , Piridinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reconocimiento en Psicología/fisiología , Reflejo de Sobresalto/fisiología , Natación/psicologíaRESUMEN
Retinal gene therapy based on adeno-associated viral (AAV) vectors is safe and efficient in humans. The low intrinsic DNA transfer capacity of AAV has been expanded by dual vectors where a large expression cassette is split in two halves independently packaged in two AAV vectors. Dual AAV transduction efficiency, however, is greatly reduced compared to that obtained with a single vector. As AAV intracellular trafficking and processing are negatively affected by phosphorylation, this study set to identify kinase inhibitors that can increase dual AAV vector transduction. By high-throughput screening of a kinase inhibitors library, three compounds were identified that increase AAV transduction in vitro, one of which has a higher effect on dual than on single AAV vectors. Importantly, the transduction enhancement is exerted on various AAV serotypes and is not transgene dependent. As kinase inhibitors are promiscuous, siRNA-mediated silencing of targeted kinases was performed, and AURKA and B, PLK1, and PTK2 were among those involved in the increase of AAV transduction levels. The study shows that kinase inhibitor administration reduces AAV serotype 2 (AAV2) capsid phosphorylation and increases the activity of DNA-repair pathways involved in AAV DNA processing. Importantly, the kinase inhibitor PF-00562271 improves dual AAV8 transduction in photoreceptors following sub-retinal delivery in mice. The study identifies kinase inhibitors that increase dual and single AAV transduction by modulating AAV entry and post-entry steps.
Asunto(s)
Terapia Genética , Vectores Genéticos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Retina/metabolismo , Transducción Genética , Animales , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Dependovirus/genética , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/genética , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/uso terapéutico , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/virología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Retina/patología , Retina/virología , Quinasa Tipo Polo 1RESUMEN
Gene therapy, in its current configuration, is irreversible and does not allow control over transgene expression in case of side effects. Only few regulated vector systems are available, and none of these has reached clinical applicability yet. The mifepristone (Mfp)-regulated Gene Switch (GS) system is characterized by promising features such as being composed of mainly human components and an approved small-molecule drug as an inducer. However, it has not yet been evaluated in adeno-associated virus (AAV) vectors, neither has it been tested for applicability in viral vectors in the central nervous system (CNS). Here, we demonstrate that the GS system can be used successfully in AAV vectors in the brain, and that short-term induced glial cell line-derived neurotrophic factor (GDNF) expression prevented neurodegeneration in a rodent model of Parkinson's disease (PD). We also demonstrate repeated responsiveness to the inducer Mfp and absence of immunological tissue reactions in the rat brain. Human equivalent dosages of Mfp used in this study were lower than those used safely for treatment of psychiatric threats, indicating that the inducer could be safely applied in patients. Our results suggest that the GS system in AAV vectors is well suited for further development towards clinical applicability.Molecular Therapy-Nucleic Acids (2013) 2, e106; doi:10.1038/mtna.2013.35; published online 16 July 2013.