CRISPR/nCas9-Edited CD34+ Cells Rescue Mucopolysaccharidosis IVA Fibroblasts Phenotype

Area of Science:

• Biochemistry
• Genetics
• Cell Biology

Background:

• Mucopolysaccharidosis (MPS) IVA is a rare genetic disorder affecting bone development, caused by a deficiency in the GALNS enzyme.
• This deficiency leads to the accumulation of glycosaminoglycans (GAGs), specifically keratan sulfate (KS) and chondroitin 6-sulfate (C6S).
• Previous research established a CRISPR/nCas9 gene therapy approach for MPS IVA in cell and animal models.

Purpose of the Study:

• To evaluate the efficacy of CRISPR/nCas9 gene therapy in editing human CD34+ cells.
• To assess the potential of these edited CD34+ cells to correct MPS IVA fibroblasts through cross-correction.
• To investigate the impact of gene editing on CD34+ cell stemness and GALNS enzyme levels.

Main Methods:

• Human CD34+ cells were electroporated with a CRISPR/nCas9 system targeting the AAVS1 locus.
• On-target insertion of a donor template and the stemness of edited CD34+ cells were evaluated.
• MPS IVA fibroblasts were co-cultured with CRISPR/nCas9-edited CD34+ cells to assess cross-correction effects.

Main Results:

• CRISPR/nCas9 gene editing effectively edited CD34+ cells without compromising their stemness.
• Edited CD34+ cells exhibited supraphysiological levels of the GALNS enzyme.
• Co-culture with edited CD34+ cells significantly increased GALNS activity in MPS IVA fibroblasts, reducing lysosomal mass and improving cellular profiles.

Conclusions:

• CRISPR/nCas9-based gene therapy can successfully edit CD34+ cells for potential MPS IVA treatment.
• Edited CD34+ cells demonstrate cross-correction capabilities, restoring GALNS enzyme activity in affected cells.
• This approach shows promise for ameliorating cellular pathologies associated with MPS IVA.