A New Gene Therapy Approach for Cardiovascular Disease by Non-coding RNAs Acting in the Nucleus.

This review discusses recent developments in the use of non-coding RNAs (ncRNAs) for the regulation of therapeutically relevant genes, with special focus on applications for the treatment of cardiovascular diseases. The interest in using ncRNAs as therapeutics has steadily increased since the discovery of RNA interference. During the last decade it has become evident that these RNAs, delivered either as oligos or expressed as small hairpin RNAs (shRNAs) from vectors, can either upregulate (transcriptional gene activation, TGA) or downregulate (transcriptional gene silencing, TGS) gene expression, typically inducing epigenetic changes in their target sites in the chromatin. Also, the important role of naturally occurring long non-coding RNAs (lncRNAs) has been recently discovered and will likely provide new insights into cardiovascular pathology and provide new treatment strategies based on the manipulation of their expression. In this review, we discuss the possibility of using ncRNAs for activating or silencing therapeutically relevant genes, such as VEGF-A, for the treatment of cardiovascular disease.

Lentiviral protein transduction with genome-modifying HIV-1 integrase-I-PpoI fusion proteins: studies on specificity and cytotoxicity.

Rare-cutting endonucleases, such as the I-PpoI, can be used for the induction of double strand breaks (DSBs) in genome editing and targeted integration based on homologous recombination. For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques. For its applications, the endonuclease needs to be transported into the target cell nucleus, where the mechanism of transport may affect its function. Here, we have studied the lentiviral protein transduction of the integrase (IN)-PpoI fusion protein using the cis-packaging method. In genome-wide interaction studies, IN-fusion proteins were verified to bind their target sequence containing 28S ribosomal RNA (rRNA) genes with a 100-fold enrichment, despite the well-documented behavior of IN to be tethered into various genomic areas by host-cell factors. In addition, to estimate the applicability of the method, DSB-induced cytotoxic effects with different vector endonuclease configurations were studied in a panel of cells. Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA. In cell studies, certain cancerous cell lines were especially prone to DSBs in rRNA genes, which led us to test the protein transduction in a tumour environment in an in vivo study. In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.

Epigenetic upregulation of endogenous VEGF-A reduces myocardial infarct size in mice.

"Epigenetherapy" alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral in vivo delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed in vivo that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity. In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes.

Epigenetherapy, a new concept.

Small RNAs have been shown to regulate gene transcription by interacting with the promoter region and modifying the histone code. The exact mechanism of function is still unclear but the feasibility to activate or repress endogenous gene expression with small RNA molecules has already been demonstrated in vitro and in vivo. In traditional gene therapy non-mutated or otherwise useful genes are inserted into patient's cells to treat a disease. In epigenetherapy the action of small RNAs is utilized by delivering only the small RNAs to patient's cells where they then regulate gene expression by epigenetic mechanisms. This method could be widely useful not only for basic research but also for clinical applications of small RNAs.

Primary effect of 1α,25(OH)2D3 on IL-10 expression in monocytes is short-term down-regulation.

The biologically most active vitamin D compound, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), influences the status of inflammation by modulating the expression of several cytokine genes. In this study, we have examined the mechanism of transcriptional regulation of interleukin 10 (IL-10) by 1α,25(OH)2D3 in lipopolysaccharide (LPS)-treated human monocytes (THP-1). Quantitative PCR showed that IL-10 mRNA expression was significantly down-regulated (2.8-fold) during the first 8h of 1α,25(OH)2D3 treatment, while after 48 h it was up-regulated (3-fold). Gel shift and quantitative chromatin immunoprecipitation (ChIP) assays showed that the vitamin D receptor (VDR) binds in a cyclical fashion to a promoter region 1500-1700 bp upstream of the IL-10 transcription start site (TSS) containing two conserved VDR binding sites. Targeting of VDR binding sites by enhancer specific duplex RNAs revealed that only the more distal element is functional and chromosome conformation capture analysis suggested that this region loops 1α,25(OH)2D3-dependently to the TSS. Quantitative ChIP and micrococcal nuclease assays also revealed 1α,25(OH)2D3-dependent cyclical epigenetic changes and nucleosome remodeling at this promoter region. In conclusion, in LPS-treated THP-1 cells the primary effect of 1α,25(OH)2D3 on IL-10 expression is down-regulation, which is achieved via a cyclical recruitment of VDR to the promoter.