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Alpha Protein Kinase 3 Gene Therapy Restores Heart Function in Mouse and Human Models of Cardiomyopathy

ABSTRACT: Truncating variants in Alpha Kinase 3 (ALPK3) cause severe cardiomyopathy for which there is no curative treatment. Here, we established an AAV-mediated gene replacement therapy to deliver full-length human ALPK3. This approach prevented cardiomyopathy development in neonatal Alpk3 mutant mice and reversed established pathology in adults. A deep proteomic interrogation demonstrated a reversal of more than 95% of the molecular signature of disease. Beyond ALPK3-deficient cardiomyopathy, we explored broader therapeutic potential based on ALPK3's regulatory role in proteostasis, a pathway commonly disrupted across cardiomyopathies. ALPK3 expression is dysregulated in cardiomyocytes harbouring titin truncating variants (TTNtv), which represent the most prevalent cause of dilated cardiomyopathy and share a common protein quality control network co-ordinated by ALPK3. Strikingly, AAV-ALPK3 completely restored contractile function in human cardiac organoids harbouring either ALPK3tv or TTNtv variants, demonstrating therapeutic efficacy across genetically distinct cardiomyopathies. These findings establish proof-of-concept for ALPK3 gene therapy in patients with ALPK3 cardiomyopathy while also revealing potential for indication expansion to TTNtv-associated cardiomyopathy, which is not amenable to conventional gene replacement therapy due to the extreme size of titin. These findings demonstrate the therapeutic potential of ALPK3-directed gene therapy as a potential curative treatment for patients with ALPK3 cardiomyopathy and other cardiomyopathies associated with disrupted proteostasis.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Sean Humphrey

LAB HEAD: Sean J Humphrey

PROVIDER: PXD077620 | Pride | 2026-06-25

REPOSITORIES: Pride

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			Action	DRS
	20260203-SA-20260119_James_McNamara_Mouse_ALPK3-ASTR1_proteome_862_F_WT_WT_Untreated.raw	Raw
	20260203-SA-20260119_James_McNamara_Mouse_ALPK3-ASTR1_proteome_873_M_KI_KI_AAV_CONTROL.raw	Raw
	20260203-SA-20260119_James_McNamara_Mouse_ALPK3-ASTR1_proteome_876_M_WT_WT_Untreated.raw	Raw
	20260203-SA-20260119_James_McNamara_Mouse_ALPK3-ASTR1_proteome_877_F_KI_KI_AAV_CONTROL.raw	Raw
	20260203-SA-20260119_James_McNamara_Mouse_ALPK3-ASTR1_proteome_881_M_KI_KI_TNNT2_ALPK3.raw	Raw

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Project description:Aims: Pathogenic truncating variants in the largest human protein TITIN are a leading cause of dilated cardiomyopathy. Because of the size of the gene encoding TITIN, many missense variations are identified. These are difficult to evaluate in genetic testing, as even individually rare variants are common in aggregate in normal populations. While the majority will be benign, a small subset is pathogenic, but distinction is challenging. Here, we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported TITIN A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Methods and Results: Heterozygous and homozygous mice carrying the TITIN A178D missense variant were characterised in vivo. Heterozygous mice had no detectable phenotype at any time point observed (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the fetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified down-regulation of TELETHONIN and FOUR-AND-A-HALF LIM DOMAIN 2, as well as the up-regulation of heat shock proteins and MYELOID LEUKEMIA FACTOR 1. Loss of TELETHONIN from the cardiac Z-disc was accompanied by proteasomal degradation; however, TELETHONIN also accumulated in the cytoplasm. In parallel, a proteo-toxic response was observed in the mice. Conclusions: We have shown that the TITIN A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism. Because the TITIN A178D variant abolishes binding of TELETHONIN, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of TELETHONIN by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model.

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Alpha Protein Kinase 3 Gene Therapy Restores Heart Function in Mouse and Human Models of Cardiomyopathy

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