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Novel Gene Therapy Based on the Activation of Endogenous Genes for the Treatment of Ischemia - Concepts of endogenetherapy, release of promoter pausing, promoter-targeted ncRNAs and nuclear RNAi

Periodic Reporting for period 3 - CleverGenes (Novel Gene Therapy Based on the Activation of Endogenous Genes for the Treatment of Ischemia - Concepts of endogenetherapy, release of promoter pausing, promoter-targeted ncRNAs and nuclear RNAi)

Reporting period: 2018-11-01 to 2020-04-30

CleverGenes project develops new therapies for ischemic heart disease by discovering new methods how to grow new blood vessels in ischemic heart muscle. Coronary heart disease is a major problem worldwide and even though current therapies can relieve symptoms, a significant number of patients still lack adequate treatment especially if they have already had bypass surgery and stent operations. CleverGenes project will use gene transfer techniques to turn on endogenous growth factors which are normally responsible for new blood vessel growth. State-of-the-art techniques will be used to utilize non-coding RNA structures in gene transfer vectors to achieve this goal. Methods will be tested in cell culture, mice and pig myocardial ischemia models. If constructs are successful and safe, they will be taken to phase I human clinical trial in refractory angina patients. If successful, the new treatments would have a significant impact on the society. The overall objective is to improve treatment of coronary heart disease and especially in patients who have no options left from the currently used clinical treatment options.
CleverGenes project has progressed according to the research plan during the first 2,5 years. Basic idea of turning on endogenous genes using various types on non-coding RNAs, enhancer elements and genetic constructs delivered with viruses has turned out to be successful and very doable approach.

Endogenous gene activation

In the promoter activation approach based on short hairpin nucleotides that bind specifically to desired promoter elements, we have shown that it is possible to activate both VEGF-A and VEGF-C expression in various endothelial cell lines from mouse, pig and human and also to regulate expression of VEGF receptor-1. Results have been recently summarized (Laham-Karam N et al. Antioxid Redox Signal 29:813-831, 2018.). We have also shown that endothelial cell differentiation is strongly influenced by long range interactions between inactive chromatin regions and that non-coding RNAs have significant roles in this process (Niskanen H et al. Nucleic Acids Res. 46:1724-1740, 2018.). These techniques have also revealed that so-called super enhancer elements found in endothelial cells and cardiomyocytes significantly affect the formation of transcriptionally active clusters and this would also explain at least partially the coordinated regulation of multiple genes during processes like angiogenesis.

In order to better understand transcription programs related to angiogenesis and hypoxia-induced effects, we have undertaken deep sequencing studies to clarify coding and non-coding RNA expression in endothelial cells. We have found a long non-coding antisense RNA, which is involved in the coordinated regulation of HIF-1α and HIF-2α. Also, target gene profiles of HIF-1α and HIF-2α are clearly distinct and therefore serve for different purposes in tissue survival and repair under ischemic attacks (Downes NL et al. Mol Ther. 26:1735-1745, 2018.). Additional information about important target genes and gene expression profiles, including non-coding RNAs, have been analyzed (Kaikkonen MU et al. Circulation: Cardiovascular Genetics. 10(3), 2017. Laakkonen JP et al. Angiogenesis. 20:109-124, 2017.).

New vector technology for safe harbor gene integration

Studies have progressed to develop better vectors, which could avoid random integration into potentially dangerous sites in human genome. This technology targets transgenes to specific sites in ribosomal RNA genes. The work has progressed more slowly because it involves extensive bioinformatics analysis of all genomic regions and interestingly enough, not all telomeric areas, which contain RNA genes, have been fully sequenced and characterized even in the latest version of human genome (hg19).

In vivo testing of new vectors and treatment constructs

We have further developed our pig chronic myocardial ischemia model and developed methods to utilize radiowater-PET imaging to quantify absolute blood flow in myocardium during acute and chronic ischemia (Nurro J et al. Heart 102:1716-1720, 2016.). Methods have already been utilized in the analysis of gene expression in normal, ischemic hibernating areas and infarction scar areas in our pig model combining pathological and sequencing findings to PET and MRI imaging (Kaikkonen MU et al. Circulation: Cardiovascular Genetics. 10(3), 2017.). We have found that lentiviral vectors cause only modest expression in normal and ischemic myocardium, whereas AAV-2 and AAV-8 vectors seem to be efficient in transducing cardiomyocytes (Lähteenvuo J & Ylä-Herttuala S. Hum Gene Ther. 28:1024-1032, 2017.).

Clinical testing of new proangiogenic endogenetherapy constructs

No clinical trial has been started since selection of the best possible constructs and delivery vectors is still under way. Therefore, no ethical or regulatory applications have been submitted.
CleverGenes project will develop new ways to turn on endogenous angiogenic growth factors by utilizing non-coding RNA structures, which can bind to promotors of endogenous growth factors and therefore turn on a natural angiogenic response in ischemic tissues. This is especially important for vascular endothelial growth factors, which produce several splice variants from their genes and in this way create gradient of growth factor in the ischemic tissues towards which new blood vessels migrate. These constructs are cloned in gene transfer vectors which are delivered to ischemic myocardium in pig models and ultimately in humans using a now electromechanic mapping and injection catheter system which can also be utilized in human patients. If successful, this will give a new minimal invasive way to treat severe myocardial ischemia without major operations and thus, this approach is applicable also to elderly patients who are not eligible for bypass surgery or angioplasty and stenting operations any more.