Periodic Reporting for period 4 - 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: 2020-05-01 to 2021-04-30 Summary of the context and overall objectives of the project 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. The first set of constructs have been tested in pig ischemia model but unfortunately unexpected toxicity was found with AAV2 vectors. Therefore, a new set of vectors based on AAV6 serotype have been generated and tested in pig ischemia model. New vector results look very good, but the large set of new AAV6 experiments have delayed the project so that the clinical trial will only be started after the CleverGenes project has been finished 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. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far CleverGenes project has progressed according to the research plan until the final safety and efficacy testing of the best constructs in pig chronic ischemia model. Basic idea of turning on endogenous genes using various types on non-coding RNAs, enhancer elements and genetic constructs delivered with viruses has been very successful and very doable approach. However, body reactions against the delivery vector produced surprising unexpected results and required a new set of experiments before clinical trial dossier could be completed. Endogenous gene activationIn the promoter activation approach based on short hairpin RNAs that bind specifically to desired promoter elements, we have shown that it is possible to activate both VEGF-A and VEGF-C expression in mouse, pig and human cells (Laham-Karam N et al. Antioxid Redox Signal 29:813-831, 2018.). 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.). We have also identified so-called super enhancers in endothelial cells and cardiomyocytes that significantly affect the formation of transcription clusters and this would also explain at least partially the coordinated regulation of multiple genes during processes like angiogenesis. We have also undertaken deep sequencing studies to clarify coding and non-coding RNA expression in endothelial cells and found a long non-coding antisense RNA, which is involved in the coordinated regulation of HIF-1α and HIF-2α and that target genes of HIF-1α and HIF-2α are clearly distinct and therefore serve different purposes after 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 integrationWe have developed a better vectors which can avoid random integration into potentially dangerous sites in genome which targets transgenes to specific sites in ribosomal RNA genes. The work has involved extensive bioinformatics analysis of all genomic regions. The current vector is very effective and produces over 20% targeted integration in the intended genome site (Schenkwein D et al, Mol Ther 2020).In vivo testing of new vectors and treatment constructsWe 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-6 vectors seem to be efficient in transducing cardiomyocytes (Lähteenvuo J & Ylä-Herttuala S. Hum Gene Ther. 28:1024-1032, 2017.). In the final safety and toxicology studies in pigs AAV2 vector turned out to cause significant fibrosis in the myocardium after long-term follow-up for 6 and 12 months, thus preventing us to move forward with the AAV2 vector platform. Therefor, we have changed the delivery vector to AAV6 which seems to be much better tolerated. A large set of animal experiments have now been performed to show efficacy and safety of AAV6 vectors to turn on endogenous proaniogenic genes. However, these new experiment have delayed the start of clinical phase 1 trial in refractory angina patients.Clinical testing of new proangiogenic endogenetherapy constructsNo clinical trial has been started with the original intended AAV2 vectors because of the unexpected toxicity found in preclinical pig experiments. The vector was changed to AAV6 vector platform, but due to extensive new animal experiments no ethical or regulatory applications have been submitted and no clinical trial started yet. However, the trial will be started later after the end of CleverGenes project. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) 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 have been cloned in gene transfer several AAV serotype vectors which are delivered to ischemic myocardium in pig models to test safety and efficacy in clinically highly relevant pig model. Once all required safety and efficacy tests have been completed, the treatment can be taken to human phase 1 clinical trial using a new electromechanic mapping and injection catheter system which is CE-marked for human use. 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. Gene therapy of Heart Diseases
