Hyperbranched poly(β-amino ester)s-mediated delivery of engineered Klotho mRNA rescues senescence and restores cellular homeostasis in aged and Klotho deficient iMSCs.

A decline in Klotho expression is a defining feature of aging and contributes to cellular dysfunctions. Here, we developed an engineered IVT Klotho mRNA incorporating ARCA capping. Ψ-modification and poly(A) tailing, delivered using a hyperbranched poly(β-amino ester) (HPAE)-based platform to enhance intracellular delivery and translation. Using CRISPR edited KL (-/-) iPSCs and derived iMSCs, we show that loss of KL induces a robust senescent phenotype characterized by activation of p53-p21-p16 pathways, mitochondrial depolarization, elevated ROS, and altered Ca2+ homeostasis. A single dose of an engineered KL mRNA restored its protein expression within 24 h and rapidly suppressed senescence markers at both transcript and protein levels. Mechanistically, KL mRNA treatment rejuvenated mitochondrial functions by restoring membrane potential, increasing SOD2 expression, reducing mitochondrial ROS, and normalizing Ca2+ homeostasis consistent with Klotho's known roles. Importantly, the rejuvenation response was selective; old and KL (-/-) iMSCs displayed substantial recovery. Whereas young iMSCs remained largely unaffected, indicating that KL acts as a stress responsive factor. These findings support engineered KL mRNA as a promising non-integrating transcript-based therapeutic approach for ameliorating senescence-associated cellular dysfunction. This work supports the potential of KL mRNA to restore regenerative capacity in older conditions and highlighting mRNA-based therapeutics as a promising direction for anti-aging and regenerative medicine.