AAV therapies for TNNT2-associated cardiac disorders

PROJECT SUMMARY Of the 7 million individuals in The US with heart failure (HF), ~1 in 500 have hypertrophic cardiomyopathy (HCM), while ~1 in 200 suffer from dilated cardiomyopathy (DCM). Large-scale genetic sequencing studies have shown that both HCM and DCM can be caused by pathogenic or likely-pathogenic (P/LP) genetic variants in the TNNT2 gene. This gene encodes cardiac troponin T, a regulatory protein in the sarcomere essential for the heart’s contractile function. ~115,000 individuals in the US with HCM or DCM have been identified to carry P/LP TNNT2 variants. These genetic variants disrupt the sarcomere’s calcium (Ca2+) sensitivity, causing hypercontractility in HCM or hypocontractility for DCM. The severity of these effects depends on the ratio of mutant TNNT2 to wild- type TNNT2 protein. Despite extensive research on the impact of P/LP TNNT2 variants, no treatments currently target this gene. Attempts to use small molecule drugs to address these genetic effects have been unsuccessful. To overcome this challenge, we engineered first generation adeno-associated viruses (AAVs) capable of delivering wild-type TNNT2 to cardiomyocytes. In pilot studies conducted in mice, these AAVs successfully prevented DCM in vivo for the first time. Additionally, the simultaneous knockdown of mutant mRNA significantly improved outcomes. To move toward clinical application, we propose optimizing and screening next-generation AAVs to identify a lead therapeutic candidate suitable for an investigational new drug (IND) application. This project follows a milestone-driven approach. In the R61 Phase: 1) AAV development and validation—to identify validate and screen promising AAV candidates; 2) micro RNA (miR) targeting—to design a miR to selectively suppress mutant TNNT2 mRNA; 3) preliminary efficacy testing—conduct human cell-based assays to evaluate efficacy and establish dose-response curves, and to utilize human 3D cardiac microtissue models of HCM and DCM to simulate real-life conditions and assess therapeutic effects; and 4) in vivo mouse studies—to perform short-term efficacy and dose-response studies using a mouse DCM model, while simultaneously identifying serum biomarkers to aid in the prediction of therapeutic response in vivo. R33 Phase progression will require identifying at least one AAV that restores normal contractile function with minimal toxicity. Key R33 activities include: 1) preclinical testing in DCM mice using a prevention and treatment study design; 2) comprehensive dose-response analyses across cardiac structure and function (echocardiography), single cardiomyocyte physiology and Ca2+ analyses (IonOptix); transcriptomic profiling (RNA-seq and qPCR) and serum biomarker studies (ELISA); and 3) tissue distribution and preliminary toxicity measurements (ddPCR, ELISA and histology). These studies will support intellectual property, large-animal safety testing, IND applications, and eventually, Phase I clinical trials in collaboration with our Accelerator Partner (JAX Accelerator), the NHLBI Catalyze Coordinating Center, and outside consultants. Our ultimate goal is to create the first TNNT2-targeted gene therapy for cardiomyopathy.