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University of Florida Health University of Florida
Norman Fixel Institute for
Neurological Diseases
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Investigator

significance

Pompe disease arises from mutations in the GAA gene, which impair glycogen breakdown within lysosomes. This accumulation of glycogen leads to serious symptoms like cardiomegaly, muscle weakness, and respiratory issues. This study seeks to enhance gene therapy for Pompe disease by exploring genetic modifications to accelerate glycogen breakdown. By modifying the AAV9-GAA vector with specific mutations at the H201 position, researchers aim to improve treatment efficacy and alleviate disease symptoms.

hypothesis

The hypothesis is that introducing the AAV9-GAA gene therapy vector with mutations at the Histidine 201 (H201) position—specifically Arginine (R), Lysine (K), or Tyrosine (Y)—will expedite glycogen breakdown, similar to the effect of the previously studied Leucine (L) mutation. This increased efficiency is expected to enhance therapeutic outcomes in Pompe disease patients.

aims

Aim A: Develop GAA gene variants with point mutations at the H201 position. Test these variants in various cell lines, including human cardiac (HL1), mouse myoblasts (C2C12), human hepatoma (HepG2), and mouse neuroblasts (Neuro2A), to assess expression and enzymatic activity compared to the wild-type GAA gene.

Aim B: Test the effectiveness of GAA-variant constructs in Pompe disease fibroblast cells, comparing their expression levels and enzymatic activity to the wild-type sequence with Histidine at the H201 position.

milestones

Milestone 1: Create mutated GAA gene variants with hydrophobic substitutions at H201. Validate the clones through Sanger sequencing.

Milestone 2: Test variants in multiple cell lines and measure GAA activity through assays and western blotting to determine increased activity over the control.

Milestone 3: Transfect Pompe disease fibroblast cells with GAA variants and validate enhanced breakdown of the GAA protein through western blot analysis.

Milestone 4: Measure GAA activity in transfected fibroblast cells, with the expectation that H201 variants will demonstrate higher activity than the wild-type.