New Cell-specific ERT Shows Promise in Mouse Study

Patricia Inácio, PhD avatar

by Patricia Inácio, PhD |

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ERT | Pompe Disease News | mouse study | illustration of mice in a lab

A new enzyme replacement therapy (ERT) targeting specific cell types boosts the delivery of acid alpha-glucosidase (GAA) — the enzyme missing or defective in Pompe disease patients — to muscle and heart cells, a study shows.

Compared to standard ERT, the targeted approach by Regeneron Pharmaceuticals normalized the levels of glycogen — the complex sugar molecule that builds up in Pompe patients — in a mouse model of the disease, particularly in skeletal muscles.

Also, the targeted ERT’s effectiveness was maintained when delivered as a gene therapy, which resulted in improved muscle function.

The study, “Cell type-selective targeted delivery of a recombinant lysosomal enzyme for enzyme therapies,” was published in the journal Molecular Therapy.

Pompe disease is caused by mutations in the GAA gene, which carries the instructions for making an enzyme of the same name. This enzyme breaks down glycogen — used for energy storage — within small structures inside cells called lysosomes. GAA deficiency leads to the accumulation of glycogen in several tissues, including heart, skeletal muscle and central nervous system (brain and spinal cord).

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ERT is the standard approach for Pompe disease. Given intravenously (into-the-vein) every other week, it involves therapeutic administration of a lab-made (recombinant) human version of the GAA enzyme, thereby allowing glycogen to be broken down.

However, “while ERT has been very successful in treating the cardiac manifestations of PD [Pompe disease], skeletal muscle and the central nervous system (CNS) remain minimally treated,” the researchers wrote.

This lack of effectiveness is linked to a poor ability of the therapeutic enzyme to reach skeletal muscle cells. The therapy is incorporated into cells via a protein called M6P receptor (CI-MPR), which is present at low levels in the skeletal muscle.

Now, researchers at Regeneron developed a targeted ERT as a way to improve the delivery of GAA enzyme to muscle cells. This new ERT was designed by fusing the enzyme to an antibody that binds to a protein located at the cell surface of skeletal muscle cells.

They selected two proteins, called CD63 and ITGA7, broadly present at the surface of the desired target cells and minimally produced in the liver (to where standard ERT is lost). Notably, CD63 was already known to aid the trafficking of molecules from the cell surface to lysosomes, supporting its potential for a targeted ERT.

First using CD63, they confirmed their newly targeted ERT retained a similar activity to the standard alglucosidase alfa, marketed by Sanofi Genzyme and marketed as Lumizyme in the U.S. and Myozyme in Europe.

Using cells grown in the lab, they observed that the targeted ERT was taken up by cells in a dose-dependent manner, meaning the higher the dose the more it was inside cells. Also, its internalization was independent of the M6P receptor.

The targeted ERT was seen to accumulate in lysosomes of fibroblasts — the main cells of connective tissue that provide structural support to muscle — isolated from a Pompe disease patient.

Next, they tested their targeted the new strategy against the standard ERT in a mouse model of the disease. The animals were engineered to produce the human version of the CD63 protein. Both ERTs were given intravenously at two equivalent doses — high and low — for four weeks.

Results showed that the targeted ERT was significantly better at reducing glycogen accumulation in muscle compared to the standard ERT.

To achieve sustained delivery of therapeutic enzyme without frequent infusions, the researchers used a harmless adeno-associated virus (AAV), commonly used as gene therapy delivery vehicles. AAVs were engineered to target the liver, an approach that has shown potential to enable continuous-dosing of ERT.

The team injected AAV with targeted ERT or standard GAA into a mouse model of Pompe disease — 1e10 or 1e11 viral genomes (vg) per mouse — and analyzed the glycogen content in skeletal and heart muscle.

The analysis revealed the highest dose of AAV-mediated targeted ERT reduced the levels of glycogen to nearly the same levels seen in the skeletal and heart muscles of healthy (wild-type) mice. The standard GAA cleared only about 50% of glycogen in muscles other than the heart.

In addition, the reduction in glycogen levels seen with the single-dose, AAV-mediated targeted ERT translated into better muscle function, with mice showing significant improvements in motor coordination and grip strength from two to three months after AAV delivery. In contrast, mice treated with standard GAA did now show significant differences compared to untreated animals.

However, no marked clearance in glycogen was seen in the CNS, with only a trend for a reduction seen in the spinal cord after 10 months.

Overall, “using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice both as a protein replacement therapeutic and as a gene therapy,” the investigators concluded.