Grant Awarded to Study Gene Therapy for Disorders With Severe Brain Disease

By Nancy McCann

Lysosomal storage disorders (LSDs) are rare, inherited disorders that render the body unable to rid itself of cellular waste. Think of lysosomes as the digestive system of the body’s cells. These little sacs within cells contain enzymes responsible for breaking down molecules. But in LSDs, one of these enzymes doesn’t work properly and so, over time, toxic substances build up in the lysosomes — thus the name — and cause damage to the body’s brain, skeleton, heart, and other organs. 

They are a large group of disorders, more than 50, with severe disease of the brain, many of which do not yet have specific treatments. There is no cure. 

“In most inherited diseases, a major barrier to effective treatment of the central nervous system is that pathology is present throughout the brain because the metabolic defect is present in most brain cells,” said John Wolfe, VMD, PhD, a researcher at Children’s Hospital of Philadelphia. 

With a grant from the National Institute of Neurological Disorders and Stroke, Dr. Wolfe is studying gene therapy methods to correct the brain disease of these disorders by improving gene and protein delivery to the entire brain. 

“This whole group of diseases has lesions throughout the brain,” Dr. Wolfe said, “so global distribution of the gene or gene product is required.”

Exploring Ways to Achieve More Complete Correction of the Brain

Specifically, his team is evaluating alpha-mannosidosis (AMD), a LSD that is caused by the mutations in the lysosomal enzyme structural gene, alpha-mannosidase (MANB). Individuals with this disease have intellectual disabilities, distinctive facial features (i.e., large head, low hairline, prominent forehead), and severe hearing loss. They may also experience speech impairments, delays in developing motor skills, and skeletal abnormalities such as reduced bone density and deformities of the spine. 

The researchers will be working with animal models to study a gene therapy treatment in which the transfer of a normal copy of the MANB enzyme into AMD cells results in the metabolic correction of those cells receiving the normal enzyme (gene-transduced cells). In addition, the enzymes can be secreted from the gene-corrected cells and used by neighboring cells. This biochemical mechanism, called cross-correction, has been known for decades, said Dr. Wolfe, director of the Walter Flato Goodman Center for Comparative Medical Genetics, School of Veterinary Medicine at University of Pennsylvania, and a professor of Pathobiology in Pediatrics in the Perelman School of Medicine at Penn. 

“We’ve tested this idea working with mice models over the years, and we have shown that if you introduce certain vectors into the cerebrospinal fluid (CSF), which is what baths the whole brain, you can get transduction — the vector entering at a cell and producing the normal protein,” he explained. 

By researching strategies that will attain more complete correction of the brain than what is currently known, Dr. Wolfe’s team aims to investigate the effects of alternative routes of CSF delivery of the vector and the effects of dosage escalation as it relates to disease resolution. Lastly, they are looking to determine the effectiveness of therapy when initiated at progressively more severe stages.

“Because this is a progressive, degenerative disease, the earlier you can treat and get the enzyme in there, you forestall more severe symptom development,” Dr. Wolfe explained. “You may not cure the disease, but the earlier you treat it, the better the outcome, in general. If we can treat the brain when it is still developing and plastic, we may be able to stop the pathology from progressing further.”

Alternative Routes of Cerebrospinal Fluid Delivery

The gene therapy vectors will be injected directly into the CSF, at two different sites of the brain: the lateral ventricles and the cisterna magna. Both areas are large reservoirs of CSF, but the fluid circulates differently from these two locations.

“We want to compare these routes by using reporter genes to see if the patterns of distribution to the hundreds of sub-regions of the brain are different and how it differs,” Dr. Wolfe said. “We need to look at all specialized areas of the brain, especially structures known as deep nuclei.” 

Part of the research involves figuring out which adeno-associated virus (AAV) serotype is the most efficient in delivering the corrected enzyme to be transduced by other cells. This is essential knowledge to have in order to scale-up and manufacture the large quantity of vector needed for clinical studies. 

“AAV is a very inefficient virus from a virologist’s point of view,” Dr. Wolfe said, “thus, a major goal is to select the most efficient among the many serotypes in order to reduce the total dose that must be given to a subject to achieve an effective treatment.” 

Dose Escalation Response

Dr. Wolfe will also investigate how increasing the dosage of the gene therapy treatment affects transduction and in turn affects the disease. In other words: How much is needed to have a positive effect on the disease?

The vector doesn’t have to get into every cell because of the cross-correction phenomena, Dr. Wolfe explained. But the amount that is secreted from one cell that can be taken up by another cell has a limit to how far it travels. 

“The theory is if you get evenly distributed cells that now have the corrected gene and each one is putting out enzyme that other cells can take up, overlapping spheres of cross-correction can cover the whole brain,” Dr. Wolfe said. 

LSDs have many mutations in the structural enzyme cells that result in different levels of activity of the enzymes, causing varying degrees of disease. For instance, if a severe mutation exists — no enzyme activity/complete loss of the gene — the disease is at its worst. If a slight mutation exists, the disease is less severe, because the cells have some level of enzyme activity. 

By testing the dosing of the therapy, the hope is to find an effective amount in which the enzyme is distributed to the diseased cells throughout the brain.

“We’re trying to refine the process to optimize the distribution of the vector correction to find the lowest level of virus (dose) that results in the enzyme reaching and correcting the metabolic pathway in all parts of the brain,” Dr. Wolfe explained. 

Measuring Effectiveness of Therapy

The team will be monitoring the disease progression and improvement from treatment at different stages of the disease (pre-symptomatic/post-symptomatic) by clinical neurological assessment, lifespan increases, serum and CSF analyses, and noninvasive brain imaging by magnetic resonance spectroscopy (MRS) and diffusion tensor imaging (DTI).

MRS is a sensitive magnetic resonance imaging (MRI) method that can measure the accumulated substrate that the enzyme fails to digest. It’s a noninvasive, analytical technique that can study metabolic changes in the brain. And DTI, another MRI method, can assess the tracks of white matter in the brain, which are severely compromised in AMD. 

“These are direct measurements of whether the gene therapy treatment is working in the brain,” Dr. Wolfe said. “I’m interested in being able to apply those methods to brain disease in a way that will be useful for monitoring disease. That’s what we’re really looking for.”