CRISPR/Cas9-based gene therapy for Angelman syndrome

基于 CRISPR/Cas9 的 Angelman 综合征基因疗法

• 批准号: 10011898
• 负责人: Mark J. Zylka
• 金额: $ 56.99万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10011898
• 关键词: Age; Alleles; Angelman Syndrome; Antisense Oligonucleotides; Behavioral; Binding; Biodistribution; Biology; Brain; CRISPR/Cas technology; Cells; Child; Childhood; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Dependovirus; Functional disorder; Gene Expression; Gene Transduction Agent; Genes; Genetic Transcription; Guide RNA; Human; Injections; Intervention; Introns; Knowledge; Light; Longevity; Mediating; Medical; Modeling; Mus; Mutagenesis; Mutation; Neurodevelopmental Disorder; Neurons; Phenotype; Pilot Projects; Pre-Clinical Model; Primates; RNA; Research; Site; Small Nucleolar RNA; Staphylococcus aureus; Streptococcus pyogenes; Testing; Therapeutic; Time; Topoisomerase Inhibitors; Topotecan; Toxic effect; Translating; UBE3A gene; Untranslated RNA; Variant; adeno-associated viral vector; autism onset; autism spectrum disorder; base; behavioral phenotyping; design; effective therapy; gene repression; gene therapy; in vivo; innovation; mammalian genome; mouse model; novel strategies; pre-clinical; relating to nervous system; side effect; therapeutic candidate; ubiquitin-protein ligase

PROJECT SUMMARY Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of UBE3A. UBE3A is biallelically expressed in nearly all cells of the body except in mature neurons, where the paternal allele is silenced by an extremely long non-coding RNA called UBE3A-ATS. In light of this biology, the most direct way to treat behavioral dysfunctions associated with AS is to unsilence the intact paternal UBE3A allele. CRISPR/Cas9 technology can be used to target specific regions of the mammalian genome for mutagenesis or transcriptional repression. In unpublished studies, we generated hundreds of S. pyogenes (Sp)Cas9 guide RNAs (gRNAs) that target regions throughout UBE3A-ATS. Several of these gRNAs, when transfected along with SpCas9, potently unsilenced paternal Ube3a in cultured mouse cortical neurons. Some of our most effective gRNAs targeted a region of Ube3a-ATS that is conserved between mice and humans, making it possible to translate our findings to human neurons. Here, we will test the central hypothesis that CNS-directed delivery of Cas9 and a gRNA that targets Ube3a-ATS can enduringly unsilence paternal UBE3A and treat behavioral phenotypes associated with Angelman syndrome. We will use adeno-associated virus (AAV) for delivery because it can drive gene expression for years in the brain. Pilot studies with S. aureus (Sa)Cas9, a smaller Cas9 variant, suggest that our gene therapy approach can be used to unsilence paternal Ube3a in mice for at least three months. To advance this innovative gene therapy towards the clinic, we will evaluate efficacy, on- and off-target effects, and mechanism of action of candidate therapeutic SaCas9 gRNAs that target Ube3a-ATS. We will use cultured neurons from AS model mice and AS-derived human neurons. We will package SaCas9 and an optimized gRNA into a single AAV vector, and then evaluate unsilencing efficacy and longevity for up to two years in mice, as well as biodistribution and toxicity. Lastly, we will evaluate the extent to which AAV-mediated delivery of this CRISPR/Cas9-based gene therapy treats behavioral phenotypes in AS model mice.

项目总结