A Transgenic Minipig Model of Huntington\u27s Disease

Background: Some promising treatments for Huntington\u27s 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)1–548 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 HTT mRNA and 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 sibling pairs 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

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

PLK1 is required for chromosome segregation, first polar body extrusion, and maintenance of the condensed state of chromosomes.

<p>(A) Experimental scheme. Oocytes were cultured for 6 hours in control medium, and then MG132 was added. The oocytes were incubated for 4 hours to arrest oocytes at the late metaphase I. After release from MG132, 100 nM BI2536 was added and oocytes were imaged. (B) Anaphase phenotypes after MG132 release in control and BI2536-treated oocytes. PB = the first polar body. (C) Imaging of securin-EGFP (green) and H2B-mCherry (red) after DMSO (control, top) or 100 nM BI2536 (lower panels) was added at the time of the MG132 washout. Each phenotype from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.g006" target="_blank">Fig. 6B</a> is shown on a representative image sequence. Arrows indicate lagging chromosomes. Note that none of the BI2536-treated oocytes undergoing abnormal chromosome segregation extruded the first polar body. Time after MG132 washout (h:mm). Scale bar = 30 μm. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s014" target="_blank">S7 Movie</a>. (D) Quantification of securin-EGFP destruction. Values were normalized to 1 when the imaging was started. Time relative to MG132 washout (h). The ‘BI2536 with anaphase’ curve represents BI2536-treated oocytes that underwent abnormal chromosome segregation either with or without DNA decondensation (3^rd and 4^th rows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.g007" target="_blank">Fig. 7C</a>). The ‘BI2536 w/o anaphase’ curve represents BI2536-treated oocytes that did not undergo chromosome segregation (2^nd row in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.g007" target="_blank">Fig. 7C</a>). Average and s.d. are shown (n = 23, 40). (E) Degradation rate of securin-EGFP calculated from (D). Average and s.d. are shown. ***p < 0.001.</p

PLK1 localizes to MTOCs and kinetochores and becomes activated around NEBD.

<p>(A) Imaging of meiosis I in oocytes expressing EGFP-PLK1 (green) and 3mCherry-CENP-C (kinetochores, red). Maximum intensity z-projection images are shown. At 7:50, the saturated signal locates at the spindle midzone, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s001" target="_blank">S1A Fig.</a> Time after induction of meiotic resumption (h:mm). Scale bar = 10 μm. Insets show magnified images on kinetochores. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s008" target="_blank">S1 Movie</a>. (B) Immunostaining of active PLK1 phosphorylated on T210 (pPLK1) during meiosis I. Pictures represent single selected confocal sections through MTOCs for pPLK1 (fire-pseudocolored or green) and pericentrin (MTOCs, red) and maximum intensity z-projection for DAPI (DNA, blue). Arrowheads indicate pPLK1 signals on MTOCs. Time after induction of meiotic resumption (h:mm). Quantification of MTOC-associated pPLK1 signals (n = 11, 5, 7, 5, 5, 9, 17 oocytes). Averages with the 95% confidence intervals are shown. Scale bar = 20 μm. Insets show magnified images on MTOCs. (C) Localization of pPLK1 on kinetochores in metaphase I oocytes. Oocytes stained for pPLK1 (green), kinetochores (CREST, red), acetylated α-tubulin, and DAPI (DNA). Scale bar = 10 μm.</p

PLK1 is required for EMI1 destruction and full APC/C activation.

<p>(A) Imaging of oocytes expressing EGFP-EMI1 or EGFP-EMI1–2A (green) and H2B-mCherry (red) in the presence of DMSO (control) or 100 nM BI2536. Scale bar = 10 μm. Time after NEBD (h). (B) Cytoplasmic EGFP-EMI1 signals were measured and normalized. Average and s.d. are shown (n = 7, 5, 3). Time after NEBD (h). (C) Imaging of securin-EGFP (green) and H2B-mCherry (red) after DMSO (control, top) or 100 nM BI2536 (bottom) was added at the time of metaphase I (6.5 hours after the induction of meiotic resumption). Note that in the control oocyte at 0:30, the metaphase plate is viewed from the top of the spindle. Time after BI2536 addition (h:mm). Scale bar = 20 μm. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s013" target="_blank">S6 Movie</a>. (D) Quantification of securin-EGFP destruction. Values were normalized to 1 at the time when imaging was started (n = 11, 18). Time is relative to BI2536 addition (h). (E) Degradation rate of securin-EGFP in oocytes with BI2536 addition during metaphase I. Average and s.d. are shown.***p < 0.001.</p