A Novel miniALPK3 Gene Therapy for ALPK3-Associated Cardiomyopathy

ALPK3 (alpha protein kinase 3) is characterized by its unique alpha kinase domain and is localized to the M band in cardiomyocytes to maintain sarcomere integrity, functioning as a pseudokinase1,2. Individuals harboring homozygous or compound heterozygous loss-of-function ALPK3 mutations present severe cardiomyopathy at birth, or in early childhood3. Heterozygous ALPK3 variants are associated with adult hypertrophic cardiomyopathy4. Consequently, restoring ALPK3 expression emerges as a promising approach for treating cardiomyopathy resulting from ALPK3 deficiency.

In exploring therapeutic strategies, we attempted to use the adeno-associated virus (AAV) vector to deliver full-length Alpk3 cDNA to cardiomyocytes in global Alpk3 knockout (Alpk3−/−) mice. These mice recapitulate the clinical manifestations of cardiomyopathies in human patients with homozygous loss-of-function ALPK3 mutations and provide a reliable model for studying ALPK3-associated cardiomyopathies1,4. All animal procedures adhered to institutional guidelines and received approval from the Institutional Animal Care and Use Committee (IACUC). However, the full-length Alpk3 coding region spans 5,040 bp, which exceeds the AAV packaging limit of 4.7 kb. ALPK3 is encoded by 14 exons, with exon 6 alone contributing to 40% of the coding sequence. Given that this region is predicted to be intrinsically disordered by in silico disorder prediction tools, we sought to generate a truncated version of ALPK3 through an in-frame deletion of a large portion of exon 6, with the goal of preserving protein functionality. Using CRISPR-Cas9, we successfully removed a 1,119 bp segment from exon 6 to create a miniALPK3 variant (Figure A). Sequencing confirmed the deletion of amino acids in exon 6 (Figure B). Mice homozygous for miniALPK3 exhibited normal cardiac function for up to 15 months of age (Figure C), demonstrating that miniALPK3 retains the functional properties of the full-length protein in the heart.