Development of an AAV-based microRNA gene therapy to treat Machado-Joseph disease

Spinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) is a progressiveneurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAGrepeat is translated into a prolonged polyglutamine repeat in the ataxin-3 protein and accumulateswithin inclusions, acquiring toxic properties, which results in degeneration of the cerebellum and brainstem.In the current study, a non-allele specific ATXN3 silencing approach was investigated using artificialmicroRNAs engineered to target various regions of the ATXN3 gene (miATXN3). The miATXN3candidates were screened in vitro based on their silencing efficacy on a luciferase reporter co-expressing ATXN3. The three best miATXN3 candidates were further tested for target engagement andpotential off-target activity in induced-pluripotent stem cells (iPSC) differentiated into frontal brain-like neurons and in a SCA3 knock-in mouse model. Besides a strong reduction of ATXN3 mRNA andprotein, small RNA sequencing revealed efficient guide strand processing without passenger strandsbeing produced. We used different methods to predict alteration of off-target genes upon AAV5-miATXN3 treatment and found no evidence for unwanted effects. Furthermore, we demonstrated in alarge animal model, the minipig, that intrathecal delivery of AAV5 can transduce the main areasaffected in SCA3 patients. These results proved a strong basis to move forward to investigatedistribution, efficacy and safety of AAV5-miATXN3 in large animals.</p

Population structure of Cryphonectria parasitica in the Modry Kamen region

The European chestnut was evaluated for Chestnut blight disease in the Modrý Kameň region, in 2010. A total of 1321 trees and sprouts were selected. There were 140 healthy trees, without any symptoms of Cryphonectria parasitica infection. Nearly 90% of the evaluated trees and sprouts were diseased. The health condition index (IHC) on 11 experimental plots varied from 2.31 to 4.03. Every collected sample was identified as having C. parasitica. All isolates had the orange culture morphology; all isolates were virulent. Among the isolates, 8 vc types were detected. Vc type EU-2 was dominant, it comprised 37.14% of the tested isolates. Two other vc types: EU 1 and EU 12 were frequently detected

A transgenic minipig models of Huntington&#8217;s disease

Background: Some promising treatments for Huntington\u2019s disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. Objective: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1\u2013548 under the control of human HTT promoter. Methods: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. Results: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTTmRNAand protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old siblings showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. Conclusions: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study