Inherited demyelinating peripheral neuropathies are progressive incurable diseases without effective treatment. To develop a gene therapy approach targeting myelinating Schwann cells that can be translatable, we delivered a lentiviral vector using a single lumbar intrathecal injection and a myelin-specific promoter. The human gene of interest, GJB1, which is mutated in X-linked CharcotMarie-Tooth Disease (CMT1X), was delivered intrathecally into adult Gjb1-null mice, a genetically authentic model of CMT1X that develops a demyelinating peripheral neuropathy. We obtained widespread, stable, and cell-specific expression of connexin32 in up to 50% of Schwann cells in multiple lumbar spinal roots and peripheral nerves. Behavioral and electrophysiological analysis revealed significantly improved motor performance, quadriceps muscle contractility, and sciatic nerve conduction velocities. Furthermore, treated mice exhibited reduced numbers of demyelinated and remyelinated fibers and fewer inflammatory cells in lumbar motor roots, as well as in the femoral motor and sciatic nerves. This study demonstrates that a single intrathecal lentiviral gene delivery can lead to Schwann cell-specific expression in spinal roots extending to multiple peripheral nerves. This clinically relevant approach improves the phenotype of an inherited neuropathy mouse model and provides proof of principle for treating inherited demyelinating neuropathies.demyelinating neuropathy | Charcot-Marie-Tooth disease | connexin32 | peripheral nerve | gene therapy
This study reports the epidemiology of respiratory syncytial virus (RSV) in hospitalized children in Cyprus over three successive seasons (2010-2013) and the association between prevalent genotypes and disease severity. RSV infections had a circulation pattern from December to March. Most RSV-positive children (83%) were aged <2 years. Genotyping of RSV isolates showed that during the first winter season of the study (2010-2011), the only RSV genotype circulating was GA2 (RSV-A), followed by genotype BA (RSV-B) in the next winter season with only few sporadic cases of GA2. During the last winter season of the study (2012-2013) the newly emerged RSV genotype ON1 (RSV-A) was virtually the only circulating genotype. Children infected with genotype ON1 suffered a significantly milder illness compared to infections with genotypes GA2 and BA with a higher percentage of BA-infected children requiring oxygen. Our findings are in contrast to the majority of published reports that suggest RSV-A causes more severe illness than RSV-B. Therefore, further investigation of the association between RSV genotypes and disease severity is required, as it might affect treatment strategies in the future.
Poliovirus type 1 neurovirulence is difficult to analyze because of the 56 mutations which differentiate the neurovirulent Mahoney strain from the attenuated Sabin strain. We have isolated four neurovirulent mutants which differ from the temperature-sensitive parental Sabin 1 strain by only a few mutations, using selection for temperature resistance: mutant S137C, was isolated at 37.5°C, S138C5 was isolated at 38.5°C, and S139C6 and S139C10 were isolated at 39.5°C. All four mutants had a positive reproductive capacity at supraoptimal temperature (Rct+ phenotype). Mutant S137C1 induced paralysis in two of four cynomolgus monkeys, and the three other mutants induced paralysis in four of four monkeys. The lesion score increased from the S137C, mutant to the S139 mutants. To map the mutations associated with thermoresistance and neurovirulence, we sequenced all regions in which the Sabin 1 genome differs from the Mahoney genome. The S137C, mutant had one mutation in the 5' noncoding region and another in the 3' noncoding region. Mutant S138C. had these mutations plus another mutation in the 3D polymerase gene. The S139 mutants had three additional mutations in the capsid protein region. The mutations were located at positions at which the Sabin 1 and Mahoney genomes differ, except for the mutation in the 5' noncoding region. The noncoding-region mutations apparently confer a low degree of neurovirulence. The 3D polymerase mutation, which distinguishes S138C. and S139 mutants from S137C1, is probably responsible for the high neurovirulence of S138C5 and S139 mutants. The capsid region mutations may contribute to the neurovirulence of the S139 mutants, which was the highest among the mutants.
Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X.
Two human neuroblastoma cell lines were persistently infected with poliovirus strains of all three serotypes. In persistently infected IMR-32 cells, which were studied in greatest detail, viral antigens were present in most cells, and over a 9-month period virions were found in the medium at high titers. Persistently infected cells were resistant to superinfection by Sabin 1, 2, and 3 poliovirus but sensitive to coxsackievirus B3. The viruses recovered from persistently infected cells were studied for conservation of epitopes, host cell specificity, and temperature resistance phenotype. The antigenic site 1 carried by the major capsid protein VP1 was modified on the persistent viruses of all three serotypes. This was confirmed for one virus by sequencing the corresponding genomic region in which two mutations were detected. The titers of persistent viruses were 1-3 log10 units higher on IMR-32 cells than on nonneuronal HEp-2 cells, while parental viruses had similar titers on both lines. When thermosensitive viruses were used to initiate the infection, the persistent viruses were found to be thermoresistant at 390C. Together the results indicate that the persistent infection correlated with the selection of highly mutated viral strains. Poliovirus-infected neuroblastoma cell lines thus constitute an in vitro model ofchronic viral infections, which are increasingly implicated in human neural diseases.Poliomyelitis paralyses are caused by poliovirus (PV)-induced necroses of motor neurons (1, 2). A late postpolio syndrome has been described: new focal motor neuron deterioration emerges after an average period of 30 years from initial infection (3, 4). One of the hypotheses proposed to account for this syndrome is a persistent viral infection (3, 4). Several members ofthe picornavirus family are effectively able to induce a persistent infection ofcells in vivo and in vitro (5-11). It is assumed that the selection of viral mutants, viral interference, and interferons play a role in this persistence (12,13). For PV, the viral cytolytic action was shown to be limited by the intercellular matrix (14). Cultures in which only a small fraction of cells were susceptible to PV have been described (15)(16)(17). In a more recent study, Kaplan et al. (18) isolated PV-infected HeLa cell lines blocked at different steps of the virus life cycle which underwent periodic crises with cytopathic effects (CPE).In cell lines of neuronal origin, PV-cell interactions have been studied only in one-step growth experiments during the first 8 hr post infection (p.i.) (19). We report here the establishment of a persistent PV infection of human neuroblastoma cells. Most cells harbored viral antigens and cultures produced virions for months at high titers in the absence of detectable CPE. Persistently infected cell lines and the resulting PV mutants are described.MATERIALS AND METHODS Materials. The Leon 37, vFG68 (20), LSc2ab (S1), P712 Ch 2ab (S2), and P3/Leon 12alb (S3) Sabin strains (21) of PV were used. Another enterovirus, coxsacki...
Gene delivery using a lentiviral vector leads to efficient gene expression specifically in Schwann cells. Restoration of Cx32 expression ameliorates nerve pathology in a disease model and provides a promising approach for future treatments of CMT1X and other inherited neuropathies.
Gap junction beta-1 (GJB1) gene mutations affecting the gap junction protein connexin32 (Cx32) cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a common inherited neuropathy. Targeted expression of virally delivered Cx32 in Schwann cells following intrathecal injection of lentiviral vectors in the Cx32 knockout (KO) mouse model of the disease has led to morphological and functional improvement. To examine whether this approach could be effective in CMT1X patients expressing different Cx32 mutants, we treated transgenic Cx32 KO mice expressing the T55I, R75W or N175D CMT1X mutations. All three mutants were localized in the perinuclear compartment of myelinating Schwann cells consistent with retention in the ER (T55I) or Golgi (R75W, N175D) and loss of physiological expression in the non-compact myelin. Following intrathecal delivery of the GJB1 gene we detected the virally delivered wild-type (WT) Cx32 in non-compact myelin of T55I KO mice, but only rarely in N175D KO or R75W KO mice, suggesting dominant-negative effects of the R75W and N175D mutants but not of the T55I mutant on co-expressed WT Cx32. GJB1 treated T55I KO mice showed improved motor performance, lower ratios of abnormally myelinated fibers and reduction of inflammatory cells in spinal roots and peripheral nerves compared with mock-treated littermates. Either partial (N175D KO) or no (R75W KO) improvement was observed in the other two mutant lines. Thus, certain CMT1X mutants may interfere with gene addition therapy for CMT1X. Whereas gene addition can be used for non-interfering CMT1X mutations, further studies will be needed to develop treatments for patients harboring interfering mutations.
Human enteroviruses are responsible for a wide spectrum of clinical diseases affecting many different organ systems. Although infection is usually asymptomatic, infections of the central nervous system manifested as meningitis or encephalitis can pose a serious public health problem, especially during outbreaks. In this study, samples from 218 patients diagnosed with enteroviral meningitis between January 2000 and December 2002 were analysed in order to assess the epidemiology of human enteroviruses as a cause of viral meningitis in Cyprus. A new typing strategy, based on partial sequencing of the 59 non-coding region (59NCR), prediction of type, and selection of type-specific primers for sensitive VP1 PCR amplification, was developed. As clustering in the 59NCR was concordant with clustering in the VP1 region, quick and reliable typing by VP1 sequencing was achieved without virus isolation in cell culture. The most frequent enterovirus serotypes identified were Human echovirus 30 (55?5 %), Human echovirus 13 (15?1 %), Human echovirus 6 (13?8 %) and Human echovirus 9 (8?3 %). Human coxsackieviruses B2, B1 and B5, Human echovirus 4, Human enterovirus 71 and Human coxsackievirus A6 represented rather rare serotypes. This is the first molecular epidemiological study of enterovirus meningitis in Cyprus. Serotype distribution corresponded basically with observations in other European countries, suggesting the spread of enteroviruses by tourism.
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