Human GLB1 knockout cerebral organoids: A model system for testing AAV9-mediated GLB1 gene therapy for reducing GM1 ganglioside storage in GM1 gangliosidosis

Abstract

GM1 gangliosidosis is an autosomal recessive neurodegenerative disorder caused by the deficiency of lysosomal β-galactosidase (β-gal) and resulting in accumulation of GM1 ganglioside. The disease spectrum ranges from infantile to late onset and is uniformly fatal, with no effective therapy currently available. Although animal models have been useful for understanding disease pathogenesis and exploring therapeutic targets, no relevant human central nervous system (CNS) model system has been available to study its early pathogenic events or test therapies. To develop a model of human GM1 gangliosidosis in the CNS, we employed CRISPR/Cas9 genome editing to target GLB1 exons 2 and 6, common sites for mutations in patients, to create isogenic induced pluripotent stem (iPS) cell lines with lysosomal β-gal deficiency. We screened for clones with <5% of parental cell line β-gal enzyme activity and confirmed GLB1 knockout clones using DNA sequencing. We then generated GLB1 knockout cerebral organoids from one of these GLB1 knockout iPS cell clones. Analysis of GLB1 knockout organoids in culture revealed progressive accumulation of GM1 ganglioside. GLB1 knockout organoids microinjected with AAV9-GLB1 vector showed a significant increase in β-gal activity and a significant reduction in GM1 ganglioside content compared with AAV9-GFP–injected organoids, demonstrating the efficacy of an AAV9 gene therapy-based approach in GM1 gangliosidosis. This proof-of-concept in a human cerebral organoid model completes the pre-clinical studies to advance to clinical trials using the AAV9-GLB1 vector.

Exploiting CRISPR Cas9 in Three-Dimensional Stem Cell Cultures to Model Disease

“…The DSB causes the cells to repair the break by inserting and deleting random sequences at the site of the break through an error prone repair mechanism called Non-Homologous End Joining (NHEJ) ( Shan et al, 2014 ). This process is often exploited when the CRISPR Cas9 system is used in stem cells to create different disease-specific models ( Lin et al, 2018 ; Latour et al, 2019 ; Strikoudis et al, 2019 ). In addition, through homology dependent repair (HDR) a specific gene or sequence can be inserted at the site of the break if that sequence is simultaneously introduced to the cell with the CRISPR Cas9 system ( Zhang et al, 2017 ).…”

“…There are three ways to incorporate CRISPR Cas9 to study stem cells in 3D organoids. One method relies on genome modification of cells prior to their encapsulation in a matrix ( Latour et al, 2019 ), while a second method involves introduction of a Cas9-sgRNA complex in organoids dissociated into single cells during infection and subsequent reformation of 3D structures ( O’Rourke et al, 2017 ). The third method involves direction delivery of Cas9 and sgRNA to the organoids without generating single cells ( Matano et al, 2015 ).…”

“…This was shown with a cerebral organoid disease model for GM1 ganglioside storage disease that results in extensive buildup of a glycosphingolipid ganglioside due to a non-functional cellular β-galactosidase ( Tonin et al, 2019 ). Human iPSCs were used to prepare isogenic cell lines with the disease carrying mutations in specific exons where most mutations for the disease were recorded in patients ( Latour et al, 2019 ). Following gene editing, increased levels of GM1 were observed in mutated organoids over non-mutated controls leading to GM1 ganglioside storage disease phenotypes recapitulated in gene-edited organoids.…”

Metabolism of Glycosphingolipids and Their Role in the Pathophysiology of Lysosomal Storage Disorders

“…Enzyme replacement therapy has shown promise in patients with GM1 gangliosidosis [108]. Recently, an adeno-associated virus serotype 9 (AAV9) gene therapy has been developed for GM1 gangliosidosis [109] and clinical trials for this treatment began in 2019 (GM1 Gangliosidosis Gene Therapy Trial (AAV9-GLB1), Clinical trial No. NCT03952637.…”

Human iPSC-Based Models for the Development of Therapeutics Targeting Neurodegenerative Lysosomal Storage Diseases

“…In particular, organoids derived from Sandhoff patients showed not only an impairment in pathways regulating CNS development and neuronal differentiation, but also increased cellular proliferation and organoid size (Allende et al, 2018). Interestingly, these human 3D brain models were successfully used to evaluate the efficacy of AAV-mediated GT (Table 1) (Allende et al, 2018;Latour et al, 2019), being not only a suitable model to investigate neurodevelopmental defects in a complex multicellular system mimicking the human brain architecture, but also a potential intermediate step between animal models and patients to evaluate vector tropism in a multicellular complex system mimicking human brain (Figure 1).…”