Efficacy of AAV serotypes to target Schwann cells after intrathecal and intravenous delivery.

To optimize gene delivery to myelinating Schwann cells we compared clinically relevant AAV serotypes and injection routes. AAV9 and AAVrh10 vectors expressing either EGFP or the neuropathy-associated gene GJB1/Connexin32 (Cx32) under a myelin specific promoter were injected intrathecally or intravenously in wild type and Gjb1-null mice, respectively. Vector biodistribution in lumbar roots and sciatic nerves was higher in AAVrh10 injected mice while EGFP and Cx32 expression rates and levels were similar between the two serotypes. A gradient of biodistribution away from the injection site was seen with both intrathecal and intravenous delivery, while similar expression rates were achieved despite higher vector amounts injected intravenously. Quantified immune cells in relevant tissues were similar to non-injected littermates. Overall, AAV9 and AAVrh10 efficiently transduce Schwann cells throughout the peripheral nervous system with both clinically relevant routes of administration, although AAV9 and intrathecal injection may offer a more efficient approach for treating demyelinating neuropathies.

Gene replacement therapy after neuropathy onset provides therapeutic benefit in a model of CMT1X.

X-linked Charcot-Marie-Tooth disease (CMT1X), one of the commonest forms of inherited demyelinating neuropathy, results from GJB1 gene mutations causing loss of function of the gap junction protein connexin32 (Cx32). The aim of this study was to examine whether delayed gene replacement therapy after the onset of peripheral neuropathy can provide a therapeutic benefit in the Gjb1-null/Cx32 knockout model of CMT1X. After delivery of the LV-Mpz.GJB1 lentiviral vector by a single lumbar intrathecal injection into 6-month-old Gjb1-null mice, we confirmed expression of Cx32 in lumbar roots and sciatic nerves correctly localized at the paranodal myelin areas. Gjb1-null mice treated with LV-Mpz.GJB1 compared with LV-Mpz.Egfp (mock) vector at the age of 6 months showed improved motor performance at 8 and 10 months. Furthermore, treated mice showed increased sciatic nerve conduction velocities, improvement of myelination and reduced inflammation in lumbar roots and peripheral nerves at 10 months of age, along with enhanced quadriceps muscle innervation. Plasma neurofilament light (NEFL) levels, a clinically relevant biomarker, were also ameliorated in fully treated mice. Intrathecal gene delivery after the onset of peripheral neuropathy offers a significant therapeutic benefit in this disease model, providing a proof of principle for treating patients with CMT1X at different ages.

Intrathecal gene therapy in mouse models expressing CMT1X mutations.

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.

Molecular epidemiology of rhinoviruses in Cyprus over three consecutive seasons.

Human rhinoviruses (HRVs) are widespread respiratory pathogens and a major cause of acute respiratory tract infections. The aim of this study was to investigate the molecular epidemiology of rhinovirus infections in children in Cyprus over three consecutive winter seasons. From a total of 116 rhinovirus-positive samples, 68 were sequenced in the 5'-UTR and VP4/VP2 regions. Thirty-six (52.9%) samples were identified as HRV-A and 27 (39.7%) as HRV-C, with only five (7.4%) samples belonging to the HRV-B species. Of these, a total of 46 different genotypes were identified. In the VP2/VP4 phylogenetic tree all strains clustered in three different well-defined clades, whereas the 5'-UTR tree exhibited clades with a mixed clustering of HRV-A and HRV-C strains reflecting the evolutionary history of recombination between HRV-A and HRV-C that has been observed previously. In summary, a high intra- and inter-season diversity of HRV types was observed. Despite its geographical isolation the frequency of HRV species in Cyprus is comparable to that reported in other regions of the world supporting the concept of an unrestricted global circulation. This study assesses, for the first time, the epidemiology of rhinovirus infections in Cypriot children and will be helpful to clinicians and researchers interested in the treatment and control of viral respiratory tract infections.

Molecular epidemiology of influenza A virus infection in Cyprus in four consecutive seasons (2009 pandemic-2013).

The aim of this study was to investigate the epidemiology of influenza A virus infection in Cyprus from the 2009 pandemic until 2013. Pandemic influenza A(H1N1)2009 virus infections outnumbered infections with other respiratory viruses until the end of 2009. The pandemic virus was also the prevalent influenza strain during influenza season 2010-2011; however, it was completely replaced by H3N2 subtype in the next season. During the most recent influenza season, 2012-2013, the pandemic strain was once again the only influenza A virus circulating in Cyprus. Full-length neuraminidase gene sequencing revealed mutations that had previously been identified as permissive. No significant difference in the expression of the IFN-inducible genes OAS and IFIT1 were observed. The phylogenetic analysis of the neuraminidase gene sequences revealed a picture of continuous importation of influenza strains in the island of Cyprus with local circulation playing only a minor role in determining the prevalent strain of the next influenza season.

Circulation of enteroviruses in Cyprus assessed by molecular analysis of clinical specimens and sewage isolates.

AIMS: To study the circulation of non-polio enteroviruses in the Cypriot population and assess the clinical relevance of different serotypes by the analysis of clinical specimens and environmental samples. METHODS AND RESULTS: Sewage samples were collected on a monthly basis for 2 years from all five districts of Cyprus. Enteroviruses were isolated using the VIRADEN method and typed by partial VP1 region sequencing. In addition, all enterovirus-positive clinical samples received during this 2-year period were typed, and a phylogenetic comparison of clinical and sewage samples based on the partial VP1 sequences was made. A significant difference between the most common serotypes found in sewage and clinical samples was observed. While Coxsackieviruses B constituted the most frequent serotypes in sewages, Echoviruses 30 and 18 prevailed in clinical samples. CONCLUSIONS: The phylogenetic analysis revealed that certain enterovirus strains circulate in the population over long period of time, while others are observed only sporadically and disappear quickly. For some serotypes, it was observed that several strains were cocirculating in the population but only some of them being detected also in clinical specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, for the first time, compares enteroviruses isolated from environmental samples and clinical specimens on a molecular level, which allowed for strain identification and discrimination. A more comprehensive molecular analysis of these strains will help identify factors, which determine different degrees of pathogenicity.