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A Highly Efficacious PS Gene Editing System Corrects Metabolic and Neurological Complications of Mucopolysaccharidosis Type I.

ABSTRACT: Our previous study delivered zinc finger nucleases to treat mice with mucopolysaccharidosis type I (MPS I), resulting in a phase I/II clinical trial (ClinicalTrials.gov: NCT02702115). However, in the clinical trial, the efficacy needs to be improved due to the low transgene expression level. To this end, we designed a proprietary system (PS) gene editing approach with CRISPR to insert a promoterless α-l-iduronidase (IDUA) cDNA sequence into the albumin locus of hepatocytes. In this study, adeno-associated virus 8 (AAV8) vectors delivering the PS gene editing system were injected into neonatal and adult MPS I mice. IDUA enzyme activity in the brain significantly increased, while storage levels were normalized. Neurobehavioral tests showed that treated mice had better memory and learning ability. Also, histological analysis showed efficacy reflected by the absence of foam cells in the liver and vacuolation in neuronal cells. No vector-associated toxicity or increased tumorigenesis risk was observed. Moreover, no off-target effects were detected through the unbiased genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) analysis. In summary, these results showed the safety and efficacy of the PS in treating MPS I and paved the way for clinical studies. Additionally, as a therapeutic platform, the PS has the potential to treat other lysosomal diseases.

SUBMITTER: Ou L

PROVIDER: S-EPMC7264433 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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A Highly Efficacious PS Gene Editing System Corrects Metabolic and Neurological Complications of Mucopolysaccharidosis Type I.

Ou Li L Przybilla Michael J MJ Ahlat Ozan O Kim Sarah S Overn Paula P Jarnes Jeanine J O'Sullivan M Gerard MG Whitley Chester B CB

Molecular therapy : the journal of the American Society of Gene Therapy 20200408 6

Our previous study delivered zinc finger nucleases to treat mice with mucopolysaccharidosis type I (MPS I), resulting in a phase I/II clinical trial (ClinicalTrials.gov: NCT02702115). However, in the clinical trial, the efficacy needs to be improved due to the low transgene expression level. To this end, we designed a proprietary system (PS) gene editing approach with CRISPR to insert a promoterless α-l-iduronidase (IDUA) cDNA sequence into the albumin locus of hepatocytes. In this study, adeno- ...[more]

PMID: 32278382

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Project description:RationaleAutosomal-dominant mutations in ryanodine receptor type 2 ( RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia.ObjectiveTo determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+).Methods and resultsGuide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 ( Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca2+ transients.ConclusionsAAV serotype 9-based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.

Dataset Information

A Highly Efficacious PS Gene Editing System Corrects Metabolic and Neurological Complications of Mucopolysaccharidosis Type I.

Publications

A Highly Efficacious PS Gene Editing System Corrects Metabolic and Neurological Complications of Mucopolysaccharidosis Type I.

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