Targeted Extracellular Vesicle Gene Therapy for Modulating Alpha-Synuclein Expression in Gut and Spinal Cord

The development of effective disease-modifying therapies to halt Parkinson’s disease (PD) progression is required. In a subtype of PD patients, alpha-synuclein pathology may start in the enteric nervous system (ENS) or autonomic peripheral nervous system. Consequently, strategies to decrease the expression of alpha-synuclein in the ENS will be an approach to prevent PD progression at pre-clinical stages in these patients. In the present study, we aimed to assess if anti-alpha-synuclein shRNA-minicircles (MC) delivered by RVG-extracellular vesicles (RVG-EV) could downregulate alpha-synuclein expression in the intestine and spinal cord. RVG-EV containing shRNA-MC were injected intravenously in a PD mouse model, and alpha-synuclein downregulation was evaluated by qPCR and Western blot in the cord and distal intestine. Our results confirmed the downregulation of alpha-synuclein in the intestine and spinal cord of mice treated with the therapy. We demonstrated that the treatment with anti-alpha-synuclein shRNA-MC RVG-EV after the development of pathology is effective to downregulate alpha-synuclein expression in the brain as well as in the intestine and spinal cord. Moreover, we confirmed that a multidose treatment is necessary to maintain downregulation for long-term treatments. Our results support the potential use of anti-alpha-synuclein shRNA-MC RVG-EV as a therapy to delay or halt PD pathology progression

Survey of Period Variations of Superhumps in SU UMa-Type Dwarf Novae

We systematically surveyed period variations of superhumps in SU UMa-type dwarf novae based on newly obtained data and past publications. In many systems, the evolution of superhump period are found to be composed of three distinct stages: early evolutionary stage with a longer superhump period, middle stage with systematically varying periods, final stage with a shorter, stable superhump period. During the middle stage, many systems with superhump periods less than 0.08 d show positive period derivatives. Contrary to the earlier claim, we found no clear evidence for variation of period derivatives between superoutburst of the same object. We present an interpretation that the lengthening of the superhump period is a result of outward propagation of the eccentricity wave and is limited by the radius near the tidal truncation. We interpret that late stage superhumps are rejuvenized excitation of 3:1 resonance when the superhumps in the outer disk is effectively quenched. Many of WZ Sge-type dwarf novae showed long-enduring superhumps during the post-superoutburst stage having periods longer than those during the main superoutburst. The period derivatives in WZ Sge-type dwarf novae are found to be strongly correlated with the fractional superhump excess, or consequently, mass ratio. WZ Sge-type dwarf novae with a long-lasting rebrightening or with multiple rebrightenings tend to have smaller period derivatives and are excellent candidate for the systems around or after the period minimum of evolution of cataclysmic variables (abridged).Comment: 239 pages, 225 figures, PASJ accepte

Elektrische Kenngrößen für die elektroporative Wirkstoff-Einschleusung und den DNA-Transfer in Zentrifugal-Aggregate von CHO-Zellen als Gewebemodell

Schmeer M. Elektrische Kenngrößen für die elektroporative Wirkstoff-Einschleusung und den DNA-Transfer in Zentrifugal-Aggregate von CHO-Zellen als Gewebemodell. Bielefeld (Germany): Bielefeld University; 2004.Die elektroporative Einschleusung von makromolekularen Farbstoffen in Chinese Hamster Ovary (CHO)-Zellen hoher Packungsdichte und die elektrischen Leitfähigkeitsänderungen der Zellen zeigen an, dass sich Membran-Elektroporen bilden und wieder ausheilen. Dabei zeigt sich, dass die Packungsdichte der Zellen einen entscheidenden Einfluss auf die feldinduzierte Membranporenbildung und damit auf den Transport von Ionen bzw. Makromolekülen durch die Membran hat. Aus der Analyse der gemessenen Leitfähigkeitsänderungen der Zellpellets während und nach Feldpulsapplikation lassen sich im Wesentlichen zwei Porenzustände deutlich unterschiedlicher Bildungs- und Ausheilkinetik und unterschiedlicher Transporteigenschaften ableiten. Die Nachfeld-Leitfähigkeitsänderungen, hervorgerufen durch die langlebigen P3-Poren, werden anhand zeit- und feldabhängiger Effluxkoeffizienten analysiert. Impedanzmessungen vor und nach Feldpulsapplikation liefern zusätzliche Informationen. Insbesondere bei Anordnungen, bei denen das Zellmaterial unmittelbaren Elektrodenkontakt hat, treten aufgrund von Elektrodeneffekten vermehrt irreversible Zellschädigungen auf. Die gewonnenen Erkenntnisse sind von essentieller Bedeutung für die Optimierung elektrischer Feldpulsparameter, z.B. beim klinischen Elektrotransfer bioaktiver Substanzen oder DNA in biologisches Material hoher Zelldichte (Gewebe) und der elektroporativen Zellfusion

Effect of the Compaction and the Size of DNA on the Nuclear Transfer Efficiency after Microinjection in Synchronized Cells

The nuclear transfer process is one of the critical rate-limiting processes in transgene expression. In the present study, we report on the effect of compaction and the size of the DNA molecule on nuclear transfer efficiency by microinjection. A DNA/protamine complex- or variously-sized naked DNA molecules were injected into the cytoplasm or nucleus of synchronized HeLa cells. To evaluate the nuclear transfer process, a nuclear transfer score (NT score), calculated based on transgene expression after cytoplasmic microinjection divided by that after nuclear microinjection, was employed. The compaction of DNA with protamine decreased the NT score in comparison with the injection of naked DNA when the N/P ratio was increased to >2.0. Moreover, when naked DNA was microinjected, gene expression increased in parallel with the size of the DNA in the following order: minicircle DNA (MC07.CMV-EGFP; 2257 bp) > middle-sized plasmid DNA (pBS-EGFP; 3992 bp) > conventional plasmid DNA (pcDNA3.1-EGFP; 6172 bp), while the level of gene expression was quite comparable among them when the DNAs were injected into the nucleus. The above findings suggest that the intrinsic size of the DNA molecule is a major determinant for nuclear entry and that minicircle DNA has a great advantage in nuclear transfer

Analytical tools in minicircle production

Rischmueller A, Viefhues M, Schmeer M, Baier R, Anselmetti D, Schleef M. Analytical tools in minicircle production. Human Gene Therapy. 2012;23(10):A143-A143