Discovery and Delivery of Cardiac Mitogens for Heart Regeneration

Abstract

The limited capacity of cardiomyocytes (CMs) to regenerate significantly impairs recovery of the adult heart from injury. As such, development of effective regenerative therapies for heart disease would require a multifaceted approach, including screening of CM mitogens in new animal and tissue models where therapeutic efficacy could be reliably evaluated. Accordingly, this goals of this thesis have been to identify new regulators of CM cell cycle, investigate the effects of the specific factor R-spondin 3 (RSPO3) on cardiac regeneration in vitro and in vivo, and optimize gene therapy delivery methods for cardiac applications.To allow unbiased identification and investigation of new CM cell cycle regulators, we developed an in vitro platform combining CRISPR knockout screening and cardiac tissue engineering technologies. This approach led to the identification of adenosine deaminase (ADA) knockout as an effective cardiac mitogen. Further investigation revealed that ADA knockout-induced CM cycling in engineered cardiac tissues (ECTs) was primarily driven by increased pentose phosphate pathway (PPP) activity. Inhibition of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, prevented ADA knockout-induced CM cycling. Conversely, G6PD overexpression promoted CM cycling. These findings highlight the role of metabolism in regulating CM proliferation and provide a versatile platform for discovery and validation of candidate mitogens for cardiac regeneration. Building on the theme of identifying new CM mitogens, we further adapted the Cancer Rainbow (Crainbow) mouse model for use in cardiac applications. These new transgenic mice allowed for CM-specific, fluorescently barcoded expression of RSPO3 variants revealing that wild-type RSPO3 uniquely increased cycling in both CMs and endothelial cells (ECs) in vivo. RSPO3 expression also increased CM size, CM cycling, and heart weight relative to body weight in neonatal mouse hearts. In vitro studies with human iPSC-derived CMs showed that recombinant RSPO3 promoted CM proliferation without decreasing the percentage of diploid CMs. Additionally, RSPO3 overexpression in neonatal mouse CMs via AAV-mediated transduction increased myocardial mass following cryoinjury. These results suggest the potential of RSPO3 to act as a cardiac mitogen in vitro and in vivo and provide a foundation for developing RSPO3-based gene therapy for cardiac regeneration. To optimize the efficacy of in vivo delivery of AAV vectors to the heart, we compared the efficacy of intramyocardial injection (IM), left main coronary catheter delivery (IC-LM), left main coronary catheter delivery with balloon occlusion (IC-BO), and cardiopulmonary bypass delivery (CPB) in porcine hearts. IM delivery resulted in the highest transduction efficiency but was spatially limited to sites of injection. IC-BO delivery achieved more uniform distribution throughout targeted areas and outperformed IC-LM and CPB delivery. A multi-species AAV capsid screen, including in vitro studies with neonatal rat ventricular myocytes (NRVMs) and human iPSC-derived CMs, as well as in vivo studies in mice and pigs, identified optimal AAV variants for each setting. Specifically, the engineered capsids SASTG and MyoAAV2a showed superior performance in vitro, with MyoAAV2a also demonstrating the highest cardiac transduction efficiency in mice. In the porcine model, the optimal capsid varied depending on the delivery method, influencing AAV transduction efficiency and distribution. The studies presented in this thesis advance our understanding of CM cell cycle regulation and lay the groundwork for development of novel cardiac regenerative therapies. Our CRISPR/ECT platform enabled discovery of ADA as a novel regulator of CM cell cycle, acting through metabolic changes in the pentose phosphate pathway. Investigation of RSPO3 revealed its potential as a cardiac mitogen, promoting cycling in both in vitro in human iPSC-derived CMs and in vivo mouse CMs. Lastly, our comprehensive analysis of AAV delivery methods and capsid variants across multiple species provided valuable insights for optimizing cardiac gene therapies in preclinical studies. Overall, these findings contribute to ongoing efforts in developing treatments to remuscularize injured hearts and improve outcomes in patients with ischemic heart disease.