PROJECT SUMMARY Alpha Protein Kinase 3 (ALPK3) is an atypical protein kinase belonging to the alpha (α)-kinase family. Loss-of- function mutations in ALPK3 result in human cardiomyopathy. Currently, no specific treatments are available for patients diagnosed with ALPK3 cardiomyopathy. To understand the in vivo role of ALPK3, we generated Alpk3 global knockout (gKO) mice, which displayed a sequential progression from early-onset dilated cardiomyopathy (DCM) to late-stage atypical hypertrophic cardiomyopathy (HCM). These mice accurately mimic the pathological development of cardiac abnormalities observed in humans carrying loss-of-function ALPK3 variants. In addition, we generated Alpk3 constitutive (cKO) and inducible (icKO) cardiac-specific knockout mouse models. Our data revealed that, like Alpk3 gKO mice, Alpk3 cKO mice develop early-onset DCM leading to premature lethality, suggesting that the primary cause of the cardiac phenotypes observed in Alpk3 gKO mice is due to loss of ALPK3 in cardiomyocytes. We also observed that adult Alpk3 icKO mice develop DCM and heart failure. Gene therapies offer an attractive approach for addressing a wide range of human diseases by directly targeting the underlying molecular causes. Adeno-associated virus (AAV)-based gene therapies have demonstrated remarkable efficacy in gene therapies for several cardiovascular diseases. We aimed to utilize a robust muscle-tropic adeno-associated virus (MyoAAV) vector to deliver Alpk3 cDNA into the myocardium of Alpk3 gKO mice, with the goal of treating ALPK3 cardiomyopathy. However, the coding sequence of the wild-type Alpk3 cDNA, encodes 1,608 amino acids (AAs), spanning 4,824 base pairs, which exceeds the 4.7 kb AAV packaging limit. Consequently, we developed a compact yet functional Alpk3 variant, named mini-ALPK3, designed for efficient AAV packaging and potential ALPK3 cardiomyopathy treatment. To validate the functionality of the mini-ALPK3 gene, we generated a mouse model in which mini-ALPK3 replaced the normal Alpk3 allele. Remarkably, mice homozygous for mini-ALPK3 exhibit normal cardiac function throughout adulthood, suggesting that mini-ALPK3 can fully compensate for the full-length ALPK3 in terms of heart functionality. Based on these compelling findings, we propose the hypothesis that mini-ALPK3 gene replacement therapy, delivered via MyoAAV vector, can effectively rescue cardiac phenotypes and premature mortality in Alpk3 knockout mice. Accordingly, our specific aims are: 1. To ascertain whether mini-ALPK3 gene replacement in Alpk3 knockout mice, administered on postnatal day 1 prior to the manifestation of cardiac abnormalities, can prevent cardiac phenotypes and rescue premature mortality; 2. To evaluate whether mini- ALPK3 gene replacement in Alpk3 knockout mice, administered at one month of age when DCM phenotypes become evident, can reverse DCM phenotypes; and 3. To investigate the capability of mini-ALPK3 gene replacement in Alpk3 knockout mice, adminis...