CRISPR-Mediated Base Editing of the Irish Infantile Tay-Sachs Mutation
Clinical Genetics and Therapeutics
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Primary Categories:
- Gene Therapy
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Secondary Categories:
- Gene Therapy
Introduction:
Infantile Tay-Sachs Disease (TSD) is a rare lysosomal storage disorder (LSD) caused by biallelic mutations in HEXA encoding the alpha subunit of a heterodimeric enzyme essential for the breakdown of GM2 ganglioside in lysosomes. One hundred and seventy-one reported pathogenic mutations cause decreased or absent beta-hexosaminidase A activity resulting in the accumulation of GM2 ganglioside in neurons of the central nervous system. This accumulation leads to impairment of lysosomal function and neuronal cell death. Infantile TSD has an onset of symptoms within the first 6 months of life including hypotonia and cherry red maculae, plateauing then loss of developmental milestones, seizures, and death between 5 to 7 years of age. While the high prevalence of mutations causing infantile TSD in the Ashkenazi Jewish population has long been recognized, the IVS9+1G>A base change in intron 9 of HEXA is more prevalent in the Irish population, with an estimated carrier frequency of 1 in 50 individuals. This project aims to demonstrate successful CRISPR-mediated base editing of the IVS9+1G>A mutation in primary human fibroblasts from TSD patients before moving to a humanized mouse model of this disease.
Methods:
The editing efficacy of an adenine base editor (ABE) paired with one of two single guide RNA (sgRNA) sequences was tested in two different lines of compound heterozygous fibroblasts from TSD patients carrying the IVS9+1G>A mutation. On and off-target base editing of this mutation will be confirmed by Sanger Sequencing and quantified using Next-Generation Sequencing. Successfully edited fibroblasts will be measured for beta-hexosaminidase activity. The percentages of successful base editing and beta-hexosaminidase restoration will be further confirmed in HEK293T cells that are haploid at the HEXA locus. These cells were transfected with a plasmid containing the humanized mouse HEXA to confirm a functional enzyme could be produced in vivo.
Results:
Both sgRNAs tested in two patient fibroblast lines demonstrated the successful base editing of the IVS9+1G>A mutation that persisted after several passages. At least one sgRNA paired with the ABE showed a four to seven-fold increase in beta-hexosaminidase A activity in edited fibroblast lines compared to unedited fibroblasts. One fibroblast line began to show a nearby off-target edit but showed a persistent increase in beta-hexosaminidase activity. These results were further confirmed by Western blotting.
Conclusion:
Both sgRNAs paired with the ABE show successful base-editing of the IVS9+1G>A mutation and beta-hexosaminidase restoration in TSD fibroblasts. However, these results should be confirmed in fibroblasts that are homozygous for this mutation or in the haploid HEK293T cells to validate these results. Once we have successfully shown base-editing in vitro, we could move to in vivo editing in a humanized mouse model carrying the mutation in human intron 9 to develop gene-editing for TSD patients carrying this mutation.
Infantile Tay-Sachs Disease (TSD) is a rare lysosomal storage disorder (LSD) caused by biallelic mutations in HEXA encoding the alpha subunit of a heterodimeric enzyme essential for the breakdown of GM2 ganglioside in lysosomes. One hundred and seventy-one reported pathogenic mutations cause decreased or absent beta-hexosaminidase A activity resulting in the accumulation of GM2 ganglioside in neurons of the central nervous system. This accumulation leads to impairment of lysosomal function and neuronal cell death. Infantile TSD has an onset of symptoms within the first 6 months of life including hypotonia and cherry red maculae, plateauing then loss of developmental milestones, seizures, and death between 5 to 7 years of age. While the high prevalence of mutations causing infantile TSD in the Ashkenazi Jewish population has long been recognized, the IVS9+1G>A base change in intron 9 of HEXA is more prevalent in the Irish population, with an estimated carrier frequency of 1 in 50 individuals. This project aims to demonstrate successful CRISPR-mediated base editing of the IVS9+1G>A mutation in primary human fibroblasts from TSD patients before moving to a humanized mouse model of this disease.
Methods:
The editing efficacy of an adenine base editor (ABE) paired with one of two single guide RNA (sgRNA) sequences was tested in two different lines of compound heterozygous fibroblasts from TSD patients carrying the IVS9+1G>A mutation. On and off-target base editing of this mutation will be confirmed by Sanger Sequencing and quantified using Next-Generation Sequencing. Successfully edited fibroblasts will be measured for beta-hexosaminidase activity. The percentages of successful base editing and beta-hexosaminidase restoration will be further confirmed in HEK293T cells that are haploid at the HEXA locus. These cells were transfected with a plasmid containing the humanized mouse HEXA to confirm a functional enzyme could be produced in vivo.
Results:
Both sgRNAs tested in two patient fibroblast lines demonstrated the successful base editing of the IVS9+1G>A mutation that persisted after several passages. At least one sgRNA paired with the ABE showed a four to seven-fold increase in beta-hexosaminidase A activity in edited fibroblast lines compared to unedited fibroblasts. One fibroblast line began to show a nearby off-target edit but showed a persistent increase in beta-hexosaminidase activity. These results were further confirmed by Western blotting.
Conclusion:
Both sgRNAs paired with the ABE show successful base-editing of the IVS9+1G>A mutation and beta-hexosaminidase restoration in TSD fibroblasts. However, these results should be confirmed in fibroblasts that are homozygous for this mutation or in the haploid HEK293T cells to validate these results. Once we have successfully shown base-editing in vitro, we could move to in vivo editing in a humanized mouse model carrying the mutation in human intron 9 to develop gene-editing for TSD patients carrying this mutation.