Genome editing in kidney organoids reveals that canonical NFκB signaling governs tubular senescence and mediates a cytotoxic and immunogenic response to AAV

Abstract

Genome editing holds transformative potential for curing human disease. However, achieving safe and effective in vivo genome editing requires further preclinical studies, to minimize risks such as genotoxicity, cytotoxicity, and immunogenicity. Unlike traditional therapies, genome editing involves irreversible DNA alterations, presenting unique challenges. This definitive and species-specific intervention necessitates additional drug development steps, ideally through human tissue models that recapitulate human-specific responses. The kidney is a predominant site for off-target effects of systemic therapies, owing to high blood flow and its concentration of exogenous factors. However, there are currently no established human models for assessing gene therapies. Here, we leverage kidney organoids as a pre-clinical testing platform for CRISPR/Cas9 genome editing via adenoassociated virus (AAV) delivery, a common strategy in clinical trials that have raised safety concerns due to supraphysiologic viral titers met with toxicity. Kidney organoids enabled the assessment of AAV tropism, revealing high transduction efficiency of AAV2 to tubules. AAV transduction, however, drove a nephrotoxic response of tubular genotoxicity, cytotoxicity, and immunogenicity. Notably, tubular injury activated IL-1β and the canonical NFκB pathway, and its targeted inhibition with bardoxolone prevented the fibrotic loss of nephrons and tubular senescence. Our findings highlight the potential of kidney organoids as a preclinical platform for gene therapeutic products, which could inform clinical trials to minimize organ failures and death in patients