1. Molecular Therapy - Nucleic Acids
Normalization of Overexpressed α-Synuclein Causing Parkinson's Disease By a Moderate Gene Silencing With RNA Interference 
Masaki Takahashi, Mari Suzuki, Masashi Fukuoka, Nobuhiro Fujikake, Shoko Watanabe, Miho Murata, Keiji Wada, Yoshitaka Nagai, Hirohiko Hohjoh 
Molecular Therapy - Nucleic Acids 
Volume 4, (January 2015)
DOI: /mtna
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Screening of siRNAs conferring a moderate level of gene silencing against SNCA. (a) Assessment of designed siRNAs. Reporter (Renilla luciferase) genes carrying two distinct SNCA target sequences, referred to as T1 and T2, were constructed using the psiCHECK-2 vector, in which a control reporter (Photinus luciferase) gene is also encoded; and siRNAs against T1 and T2 were designed and assessed. Briefly, the constructed target reporter plasmid and a test siRNA were cotransfected into HeLa cells, and 24 hours after transfection, luciferase activities were examined. Modified (mismatched) siRNAs are indicated by #. Normalized data with control reporter activities were further normalized to the data obtained with a nonsilencing siRNA, siControl, as 1. Data are shown as mean + SD. (n = 4). Approximately 40–60% of the expression of target reporter genes is indicated in gray. (b) IC50 analysis. The siSNCA_T1-2(10U) and siSNCA_T2-9 siRNAs, which showed a moderate level of gene silencing in a, were further examined by IC50 analysis. In addition, siSNCA_T1-2 and siSNCA_T2-11, both of which conferred a strong gene silencing, were also examined as controls. Data are shown as mean ± SD (n = 4).
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Moderate gene silencing against endogenous SNCA. The siSNCA_T1-2(10U) and siSNCA_T2-9 siRNAs at 10–1, 100, and 101 nmol/l final concentrations were introduced into HeLa cells (a), and siSNCA_T1-2 that showed a strong gene silencing (Figure 1), was also examined as a control (b). Twenty four hours after introduction, total RNAs were prepared and the level of endogenous SNCA was examined by RT-qPCR using GAPDH as an internal reference. Normalized expression data were further normalized to the data obtained with 101 nmol/l siControl giving as 1. Data are shown as mean + SD (n = 4).
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Expression-control RNAi against overexpressed SNCA in PD patient's fibroblasts. (a) Endogenous SNCA level after RNAi treatment. The siSNCA_T2-9 and siControl (as a control) were introduced into PD patient's fibroblast possessing SNCA locus triplication by using a ViaFect transfection reagent (Promega) (see Supplementary Figure S4 for its transfection efficiency). To see a normal SNCA expression level, healthy individual's fibroblasts (normal) that were transfected with siControl (siCont) were also investigated. Twenty-four hours after transfection, total RNAs were prepared from cells and examined by RT-qPCR using the SNCA and GAPDH primers as in Figure 2. Normalized expression data were further normalized to the data obtained from PD fibroblasts that were treated with siControl, giving as 1. Data are shown as mean + SD (n = 3). (b) Gene expression profile analysis. The total RNAs that were extracted from the siSNCA_T2-9- and siControl-treated PD fibroblasts (the same samples as in a) were further subjected to a gene expression analysis using DNA microarrays. The difference in gene expression profile between the treated PD fibroblasts was investigated by a scatter-plot graph. The equal level of gene expression between the two samples is indicated by a red line, and twofold differences (increase and decrease) in the level of gene expression between them are indicated by blue lines. Note that little or no significant difference in the expression level of genes except lower-expressed genes (<102) between the samples were observed.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Screening siRNAs and shRNAs in Drosophila S2 cells. (a) Assessment of synthetic siRNAs in S2 cells. The synthetic siRNAs, which were examined in HeLa cells (Figures 1 and 2), were further examined in S2 cells where pAc-Renilla-SNCA_T1 and pAc-Photinus encoding the T1 target sequence and a control (Photinus luciferase) reporter, respectively, were cotransfected. The expression levels of the reporter genes were examined as in Figure 1. Data are shown as mean + SD (n = 4). (b) Assessment of shRNA expression plasmids in S2 cells. Based on siSNCA_T1-2(10U), shRNA expression plasmids (indicated) were constructed, and assessed in S2 cells as in a. Normalized data were further normalized to the data obtained with pshControl plasmid encoding a non-silencing shRNA, giving as 1. The pshSNCA_T1-2(Long) plasmid (indicated by #) produces a slightly long hairpin RNA relative to the shRNA of pshSNCA_T1-2. Data are shown as mean + SD (n = 6).
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Expression control RNAi (ExCont-RNAi) in PD-model flies.(a) Phenotypes of generated transgenic flies (tg-flies). The generated tg-flies were put into numerical order (#1∼#6) and also discriminated in different colors. Since a pan-neuronal protein, Elav, can trigger the expression of GAL4 (elav-GAL4) in tg-flies, all the transgenes are specifically expressed in the central nervous system of the flies. (b) Western blot analysis. SNCA that was expressed in the head of indicated PD-model flies was examined at different ages after eclosion. α-tubulin as an internal reference and GFP were also examined. Note that the GFP expression means the presence of shRNAs targeting SNCA, because the 3’ untranslated region of GFP contains the sequence of shRNA. Relative SNCA level is indicated at the bottom graph: the band intensity of SNCA was normalized to that of α-tubulin, and further normalized to the value of #4 at the age of 1 week, giving as 1. (c) Age-related change in climbing-ability. Climbing assay was carried out at indicated ages after eclosion. Details are in Materials and Methods. Tg-flies examined are indicated in different colors as in a. Data are shown as mean ± SD. (10–20 individual flies/test; 10 tests/fly line). (d) End-point climbing assay. The data of the climbing-abilities of tg-flies at the age of 5 weeks after eclosion were indicated, and the examined tg-flies are indicated in different colors as in a. Data are shown as mean + SD. (10–20 individual flies/test; 10 tests/fly line). Statistical analyses were carried out using one-way analysis of variance followed by Tukey–Kramer test (*P < 0.05; n.s., no significant difference).
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions