Gene therapy for heart disease currently requires dangerously high doses of a viral delivery vehicle, called AAV, because heart muscle cells resist infection. This project will develop small RNA molecules—microRNAs, siRNAs, and anti-microRNAs—that act as biological "adjuvants" to make heart cells more receptive to both standard gene therapy and CRISPR-based gene editing. The problem is that AAV vectors work poorly on heart cells, forcing doctors to use toxic doses. Similarly, most gene editing today can only disable faulty genes in cells grown in a dish, not repair mutations inside a living heart. This project tackles both bottlenecks by targeting the cell’s own machinery rather than redesigning the virus or the editor. If successful, the approach could lower the dose of AAV needed for cardiac gene therapy, reducing side effects. It could also enable precise repair of mutations that cause hypertrophic and dilated cardiomyopathy—two common inherited heart conditions. The researchers will test their adjuvants in mice and pigs, and develop gene editing for two mouse models carrying human mutations in the *MYBPC* and *RBM20* genes. Beyond immediate treatment, the project will uncover fundamental mechanisms controlling how AAV transfers genes and how cells carry out precise DNA repair, advancing the field beyond current limits.
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Progress in gene therapy has been remarkable over the last decade. While most of the in vivo applications are based on the adenoassociated virus (AAV), broader use of these vectors is limited by their tropism for post mitotic cells and their relatively low efficiency. Very high doses are thus required, with consequent toxicity. Analogous efficiency considerations also apply to CRISPR/Cas9 gene editing. In the heart, this technology would offer the ultimate solution for many hereditary conditions. However, most gene editing applications to date focus on ex vivo cell treatment and to the introduction of mutations that inactivate a genomic element, rather than pursuing true mutation repair in vivo. How can we improve AAV gene therapy and precise gene editing? While many laboratories focus on vectors and editors, HELP-GT pursues the innovative idea to act on the target cells. We aim develop reagents that render cardiomyocytes more permissive to transduction, or to homology direct repair or prime editing. This concept is analogous to that of adjuvants in immunology. Our adjuvants are small non coding RNAs (microRNAs, siRNAs, anti-microRNA), which we identify through systematic screenings and then formulate using lipid nanoparticles together with AAV. For screening and testing, we have available platforms based on primary rodent cardiomyocytes, human cardiomyocytes from iPS cells and human myocardial slices from explanted hearts. For gene therapy, we will test our small RNA adjuvants in both mice and pigs, also to identify a most effective administration route. Precise gene editing will be developed for two mouse models carrying human mutations in the genes for MYBPC and RBM20, which are frequent causes of hypertrophic and dilated cardiomyopathy respectively. Besides its translational value, HELP-GT will offer an unprecedented possibility to understand the mechanisms that regulate AAV gene transfer and gene editing, and thus advance the field beyond state-of-the-art.
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