Dual-AAV gene replacement for SCN2A disorders

About This Grant

Project Summary/Abstract SCN2A encodes the voltage-gated sodium channel Nav1.2, an important channel mainly expressed in the brain to mediate neuronal action potential firing. Recent large-scale genetic studies in humans have demonstrated that mutations in SCN2A are one of the leading causes of neurodevelopmental disorders. Protein- truncating variants (PTVs), which lead to SCN2A deficiency, represent a sizable proportion. However, there is no effective treatment or cure for these genetic disorders. Our lab has established a “gene trap” Scn2a-deficient mouse model and found that partial restoration of the SCN2A gene expression can rescue key disease phenotypes. Restoration of gene expression by the exogenous supply of functional genes is a demonstrated approach to treating monogenic diseases caused by gene deficiency. Thus, developing a “gene replacement therapy” to restore SCN2A expression could be a potential therapeutic strategy. AAV is an FDA-approved delivery vehicle used in different types of gene therapies. However, one major limitation of AAV is its relatively small packing capacity. The SCN2A gene is beyond the packaging capacity of a single AAV. To overcome this packaging limitation, technologies were developed to separate a single gene into two parts for packing into two AAVs (Dual-AAV), which can then be assembled inside the cells to produce a full-length functional protein. To test the SCN2A replacement strategy in both mouse models and human cells, our lab has also established human induced pluripotent stem cells (hiPSCs) carrying SCN2A PTVs. We differentiated these hiPSCs into 3D brain organoids, and our preliminary study uncovered unique human cell-specific phenotypes, highlighting the utility of human cell-based models. In this application, we propose to test an overarching hypothesis that delivery of functional WT SCN2A via Dual-AAV vectors will rescue behavioral deficits in Scn2a-deficient mice and cellular phenotypes in human brain organoids carrying SCN2A PTVs. Our proposed study is significant because: i) It tackles one of the leading monogenic causes (SCN2A) of neurodevelopmental disorders; ii) Gene replacement is an FDA-approved therapeutic strategy for many genetic disorders. Our proposed Dual-AAV strategy will bypass the packaging limitation of AAV to deliver large genes like SCN2A to treat an expanded list of genetic disorders; iii) We will use both transgenic mouse models that show robust behavioral abnormalities and human cell-based brain organoid models that reveal human cell-specific phenotypes to comprehensively test our novel genetic interventions. By completing this study, we expect to move much closer toward gene replacement therapy for SCN2A disorders. Additionally, our study is also expected to impact other fields that benefit from Dual-AAV, including many neurological diseases caused by the deficiency of large genes.