Since iPSCs have the potential to generate virtually any other cell, including nerve cells, they can be used as cellular models of Sanfilippo type B to better understand the underlying mechanisms of the disease.
They also can be used to test potential therapies.
The research was outlined in “Generation of two NAGLU-mutated homozygous cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 to model Sanfilippo B syndrome,” a study published in the journal Stem Cell Research.
Sanfilippo syndrome type B — also known as mucopolysaccharidosis type IIIB — is an early neurodegenerative disorder caused by mutations in the NAGLU gene, which contains the instructions to make the alpha-N-acetylglucosaminidase (NAGLU) enzyme.
These mutations result in reduced NAGLU activity, leading to the toxic accumulation of long complex sugar molecules, called heparan sulfate, inside cells and neurodegeneration.
Since there are no specific therapies for Sanfilippo type B, current patient care focuses on minimizing disease symptoms. Preclinical models of the disease, therefore, are important for understanding the mechanisms behind the disease and identifying potential therapeutic targets and therapies.
Researchers at the University of Barcelona, in Spain, and at the Lund Stem Cell Center, in Sweden, have now developed two iPSC lines with mutations in both copies of the NAGLU gene, using CRISPR-Cas9 gene editing technology.
iPSCs are derived from differentiated cells — skin or blood cells — that have been reprogrammed back into a stem cell-like state, which has the potential to generate virtually any cell type in the body.
The CRISPR-Cas9 system, which is similar to the editing system used by bacteria as a defense mechanism, allows researchers to edit parts of the genome by adding, removing, or changing specific sections of DNA, just like a tailor-made genetic modification.
In this study, researchers used the CRISPR-Cas9 system to generate two different NAGLU-mutated iPSC lines from an established healthy iPSC line named UBi001-A. The resulting iPSC lines were called UBi001-A-3 and UBi001-A-4.
UBi001-A-3 had a DNA deletion in the NAGLU gene (called p.F288_I290del) that led to the loss of three amino-acids — the building blocks of proteins — in the NAGLU protein. UBi001-A-4 presented a frameshift mutation (called p.F288Ifs*27) — which changes the way a gene sequence is read — that resulted in a premature stop in protein production, leading to a shorter protein. Both mutations were likely to change NAGLU’s protein structure and affect its activity.
When comparing NAGLU’s activity between the two NAGLU-mutated cell lines and the healthy cell line, as well as between nerve cells derived from all three cell lines, the team found that both NAGLU mutations led to a clear reduction in NAGLU activity.
Further analysis also showed that both NAGLU-mutated iPSC lines maintained their ability to give rise to the three layers of tissue found in an embryo — ectoderm, mesoderm, and endoderm – from which all other tissues in the body originate.
These findings suggest that both NAGLU-mutated iPSC lines, together with the healthy iPSC line, “can be useful for disease modeling as well as to identify potential therapeutic approaches for Sanfilippo B syndrome,” the researchers said.