Efficient in vivo knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA

BACKGROUND: Adenovirus (Ad) mediated gene transfer is a well-established tool to transiently express constructs in livers of mice in vivo. In the present study, we determined the specificity and efficiency of Ad vectors expressing short hairpin (sh) RNA constructs to knock-down the estrogen receptor α (ERα). RESULTS: Two different shRNA constructs derived from the murine ERα coding sequence were designed (shERα). In vitro, transfection of three mouse cell lines with pSUPER-shERα constructs resulted in up to 80% reduction of endogenous ERα activity. A single mismatch in the target sequence eliminated the reduction of ERα activity, demonstrating the specificity of shERα. The subsequently generated Ad.shERα vectors were equally effective in vitro. In vivo, intravenous administration of Ad.shERα resulted in 70% reduced hepatic mouse ERα mRNA levels. Co-injection of Ad.shERα with an Ad vector containing a luciferase (luc) gene driven by an estrogen responsive element (ERE) containing promoter resulted in a significant (90% on day five) down-regulation of hepatic luciferase activity, as determined by non-invasive optical imaging. Down-regulation was sustained up to day seven post-injection. CONCLUSION: Ad mediated transfer of shERα expression constructs results in efficient and specific knockdown of endogenous ERα transcription both in vitro and in vivo

Downregulation of miRNA-29, -23 and -21 in urine of Duchenne muscular dystrophy patients

AIM: To study the signature of 87 urinary miRNAs in Duchenne muscular dystrophy (DMD) patients, select the most dysregulated and determine statistically significant differences in their expression between controls, ambulant (A) and nonambulant (NA) DMD patients, and patients on different corticosteroid regimens. Patients/materials & methods: Urine was collected from control (n = 20), A (n = 31) and NA (n = 23) DMD patients. miRNA expression was measured by reverse transcription-quantitative PCR. RESULTS: miR-29c-3p was significantly downregulated in A DMD patients while miR-23b-3p and miR-21-5p were significantly downregulated in NA DMD patients compared with age-matched controls. CONCLUSION: miR-29c-3p, miR-23b-3p and miR-21-5p are promising novel noninvasive biomarkers for DMD, and miR-29c-3p levels are differentially affected by different steroid regimens, supporting the antifibrotic effect of steroid therapy

T Cell Responses to Dystrophin in a Natural History Study of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, but many patients have rare revertant fibres that express dystrophin. The skeletal muscle pathology of DMD patients includes immune cell infiltration and inflammatory cascades. There are several strategies to restore dystrophin in skeletal muscles of patients, including exon skipping and gene therapy. There is some evidence that dystrophin restoration leads to a reduction in immune cells, but dystrophin epitopes expressed in revertant fibres or following genome editing, cell therapy or microdystrophin delivery after AAV gene therapy may elicit T cell production in patients. This may affect the efficacy of the therapeutic intervention, and potentially lead to serious adverse events. To confirm and extend previous studies, we performed annual Enzyme- Linked Immunospot interferon-gamma assays on peripheral blood mononuclear cells from 77 paediatric boys with DMD recruited into a natural history study, 69 of whom (89.6%) were treated with corticosteroids. T cell responses to dystrophin were quantified using a total of 368 peptides spanning the entire dystrophin protein, organized into nine peptide pools. Peptide mapping pools were used to further localize the immune response in one positive patient. Six (7.8%) patients had a T cell-mediated immune response to dystrophin at at least one timepoint. All patients that had a positive result had been treated with corticosteroids, either prednisolone or prednisone. Our results show that ~8% of DMD individuals in our cohort have a pre-existing T cell-mediated immune response to dystrophin despite steroid treatment. Although these responses are relatively low-level, this information should be considered as a useful immunological baseline before undertaking clinical trials and future DMD studies. We further highlight the importance for a robust, reproducible standard operating procedure for collecting, storing and shipping samples from multiple centres to minimise the number of inconclusive data

DUX4 induces a transcriptome more characteristic of a less-differentiated cell state and inhibits myogenesis

Neurological Motor Disorder

Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA-2

<p><b>Copyright information:</b></p><p>Taken from "Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA"</p><p>BMC Biotechnology 2006;6():11-11.</p><p>Published online 28 Feb 2006</p><p>PMCID:PMC1403768.</p><p>Copyright © 2006 Krom et al; licensee BioMed Central Ltd.</p>days after Ad. administration and subjected to taqman analysis. The cyclophillin gene was used as internal standard. Data represented as mean ± SD

Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA-0

<p><b>Copyright information:</b></p><p>Taken from "Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA"</p><p>BMC Biotechnology 2006;6():11-11.</p><p>Published online 28 Feb 2006</p><p>PMCID:PMC1403768.</p><p>Copyright © 2006 Krom et al; licensee BioMed Central Ltd.</p>hERα_1103, or pSUPER- shERα_tandem. Subsequently, the cells were treated 24 hours with 10M 17-β-estradiol. Luciferase activity was measured 48 hours after transfection and after correction for LacZ expression, represented as the mean (n = 3) ± SD relative to the transfection with pSUPER-empty. (A) Endogenous mouse ERα mediated transcription in EOMA, H5V and MXT cells after introducing pSUPER +/- shERα. (B) The 19-nt target-recognition sequence of ERα_1395 contains one mismatch with human ERα and five mismatches with the mouse ERβ sequence. (C) ERα mediated transcription in EOMA cells after over expression of either mouse ERα- or human ERα-expression vectors in presence of pSUPER empty or pSUPER shERα_1395 (D) Western blot analysis of H5V cells co-transfected with pCMV-mERα and pSUPER-empty or pSUPER-shERα_1395. The lysates were analysed by immunoblotting (insert-photo) with anti-mouse ERα and anti-p38. The intensity of the bands was quantified and normalized to cells transfected with pSUPER-empty. The relative ERα protein levels are presented (bar-diagram) as mean (n = 3) +/- SD

Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA-1

<p><b>Copyright information:</b></p><p>Taken from "Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA"</p><p>BMC Biotechnology 2006;6():11-11.</p><p>Published online 28 Feb 2006</p><p>PMCID:PMC1403768.</p><p>Copyright © 2006 Krom et al; licensee BioMed Central Ltd.</p>tudy. (B) EOMA and MXT cells were co-transfected with pERE-Luc and pCMV-LacZ and than infected either with Ad.Empty, Ad.shERα_1395, or Ad.shERα_1103. 10M Estrogen was administrated for 24 hours. Luciferase activity was measured 48 hours after infection. Data represented as mean ± SD relative to infection with Ad.Empty

Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA-3

<p><b>Copyright information:</b></p><p>Taken from "Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA"</p><p>BMC Biotechnology 2006;6():11-11.</p><p>Published online 28 Feb 2006</p><p>PMCID:PMC1403768.</p><p>Copyright © 2006 Krom et al; licensee BioMed Central Ltd.</p>cipients were treated for 6 hours with increasing amounts of estrogen (0–50 μg/kg, s.c). Then, the mice were sacrificed, and the livers were processed for luciferase assays. Luciferase activity is expressed as relative luciferase units (RLU) per mg total liver protein. (B) Male C57Bl/6 mice (n = 5) were injected with Ad.ERE-Luc (5 × 10pfu) plus Ad.Empty or Ad.shERα_1103 (3 × 10pfu). Three or seven days post-infection, the mice were injected with 5 μg/kg estrogen. The (inset) photo shows the result of optical imaging of the bioluminescence at day three, the bar-diagram is a quantitative representation of hepatic luciferase activity at day three or day seven. (C) Male C57Bl/6 mice (n = 5) were co-injected with Ad.ERELuc (5 × 10pfu) + Ad.Empty or Ad.shERα_1103 (3 × 10pfu). Five days later, the mice received 0 or 5 μg/kg estrogen. After 6 hours, the animals were sacrificed, and hepatic luciferase activity was determined. Luciferase activity is expressed as relative luciferase units (RLU) per mg total liver protein. Data represented as mean ± SD