Multiplex gene editing reduces oxalate production in primary hyperoxaluria type 1.

Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I (PH1), the most common and life-threatening type of primary hyperoxaluria. The compact Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) from the and (Cpf1) protein simplifies multiplex gene editing and allows for all-in-one adeno-associated virus (AAV) delivery. We hypothesized that the multiplex capabilities of the Cpf1 system could help minimize oxalate formation in PH1 by simultaneously targeting the hepatic hydroxyacid oxidase 1 ( ) and lactate dehydrogenase A ( ) genes. Study cohorts included treated PH1 rats ( rats injected with AAV-AsCpf1 at 7 days of age), phosphate-buffered saline (PBS)-injected PH1 rats, untreated PH1 rats, and age-matched wild-type (WT) rats. The most efficient and specific CRISPR RNA (crRNA) pairs targeting the rat and genes were initially screened . experiments demonstrated efficient genome editing of the and genes, primarily resulting in small deletions. This resulted in decreased transcription and translational expression of and . Treatment significantly reduced urine oxalate levels, reduced kidney damage, and alleviated nephrocalcinosis in rats with PH1. No liver toxicity, ex-liver genome editing, or obvious off-target effects were detected. We demonstrated the AAV-AsCpf1 system can target multiple genes and rescue the pathogenic phenotype in PH1, serving as a proof-of-concept for the development of multiplex genome editing-based gene therapy.