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 activation
In 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 integration
We 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 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-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 constructs
No 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.
