Multiplex gene editing reduces oxalate production in primary hyperoxaluria type 1

Area of Science:

• Biochemistry
• Genetics
• Molecular Biology

Background:

• Primary hyperoxaluria type I (PH1) is a severe genetic disorder characterized by excessive oxalate production, leading to kidney damage and failure.
• Current therapeutic strategies for PH1 are limited, necessitating novel approaches to reduce oxalate synthesis.

Purpose of the Study:

• To investigate the efficacy of a multiplex gene editing approach using the CRISPR-Cpf1 system to simultaneously target hepatic hydroxyacid oxidase 1 (Hao1) and lactate dehydrogenase A (Ldha) genes in a rat model of PH1.
• To assess the potential of this strategy for reducing oxalate formation and ameliorating PH1-associated pathology.

Main Methods:

• Screening of CRISPR RNA (crRNA) pairs targeting rat Hao1 and Ldha genes ex vivo for optimal efficiency and specificity.
• In vivo delivery of adeno-associated virus (AAV)-AsCpf1 to PH1 rats to achieve multiplex genome editing of Hao1 and Ldha.
• Evaluation of genome editing efficiency, gene expression changes, urine oxalate levels, kidney damage, and nephrocalcinosis.

Main Results:

• Efficient genome editing of Hao1 and Ldha genes was achieved in vivo, primarily through small deletions, leading to reduced gene expression.
• Treatment with AAV-AsCpf1 significantly decreased urine oxalate levels and alleviated kidney damage and nephrocalcinosis in PH1 rats.
• No significant liver toxicity, extrahepatic genome editing, or off-target effects were detected.

Conclusions:

• The AAV-AsCpf1 system demonstrates potent multiplex gene editing capabilities for simultaneously targeting multiple genes involved in oxalate synthesis.
• This study provides a proof-of-concept for multiplex genome editing-based gene therapy as a promising therapeutic strategy for PH1.
• The approach offers a potential avenue for developing novel treatments to manage or cure PH1 by addressing its root cause.