RESUMEN
OBJECTIVE: Temporal lobe epilepsy (TLE) is characterized by recurrent seizures generated in the limbic system, particularly in the hippocampus. In TLE, recurrent mossy fiber sprouting from dentate gyrus granule cells (DGCs) crea an aberrant epileptogenic network between DGCs which operates via ectopically expressed GluK2/GluK5-containing kainate receptors (KARs). TLE patients are often resistant to anti-seizure medications and suffer significant comorbidities; hence, there is an urgent need for novel therapies. Previously, we have shown that GluK2 knockout mice are protected from seizures. This study aims at providing evidence that downregulating KARs in the hippocampus using gene therapy reduces chronic epileptic discharges in TLE. METHODS: We combined molecular biology and electrophysiology in rodent models of TLE and in hippocampal slices surgically resected from patients with drug-resistant TLE. RESULTS: Here, we confirmed the translational potential of KAR suppression using a non-selective KAR antagonist that markedly attenuated interictal-like epileptiform discharges (IEDs) in TLE patient-derived hippocampal slices. An adeno-associated virus (AAV) serotype-9 vector expressing anti-grik2 miRNA was engineered to specifically downregulate GluK2 expression. Direct delivery of AAV9-anti grik2 miRNA into the hippocampus of TLE mice led to a marked reduction in seizure activity. Transduction of TLE patient hippocampal slices reduced levels of GluK2 protein and, most importantly, significantly reduced IEDs. INTERPRETATION: Our gene silencing strategy to knock down aberrant GluK2 expression demonstrates inhibition of chronic seizure in a mouse TLE model and IEDs in cultured slices derived from TLE patients. These results provide proof-of-concept for a gene therapy approach targeting GluK2 KARs for drug-resistant TLE patients. ANN NEUROL 2023;94:745-761.
Asunto(s)
Epilepsia Refractaria , Epilepsia del Lóbulo Temporal , MicroARNs , Humanos , Ratones , Animales , Epilepsia del Lóbulo Temporal/terapia , Lóbulo Temporal , Hipocampo , Epilepsia Refractaria/genética , Epilepsia Refractaria/terapia , ConvulsionesRESUMEN
The current treatments used against RNA viruses have a limited efficacy and are often hampered by the induction of side-effects. The specific delivery of antiviral proteins in infected cells should increase their efficiency and reduce their impact on healthy cells. Here, we describe the development of a new approach which takes advantage of the viral replication machinery to specifically target the antiviral protein expression to the infected cells. The strategy is based on the delivery of a non-coding (-)RNA carrying the structures required for the binding of the viral replication complex and the complementary sequence of an antiviral gene. The viral replication complex replicates the (-)RNA similarly to the viral genome to give a coding (+)RNA from which the antiviral protein will be expressed. As non-infected cells do not express the replication complex, this specific machinery can be used to target virus-infected cells without affecting healthy cells. We show that this approach can be successfully applied to the hepatitis C virus. In both replicon-harboring cells (genotype 1b) and JFH-1 infected cells (genotype 2a), nrRNAs induced a strong decrease in genomic RNA and viral protein NS5A. These effects were correlated with a strong activation of several interferon-stimulating genes.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/fisiología , ARN no Traducido/farmacología , Replicación Viral/efectos de los fármacos , Línea Celular , Hepatocitos/virología , Humanos , ARN Viral/biosíntesis , Proteínas no Estructurales Virales/biosíntesisRESUMEN
We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5' end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.
Asunto(s)
Regulación Viral de la Expresión Génica , Hepacivirus/genética , Biosíntesis de Proteínas , Proteínas no Estructurales Virales/genética , Regiones no Traducidas 5'/química , Línea Celular , Clonación Molecular , Biblioteca de Genes , Humanos , ARN Viral/química , Secuencias Reguladoras de Ácido RibonucleicoRESUMEN
We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.