Now researchers at Stanford Medicine have developed a way to replace more than half of the most severely affected cells, called microglia, with non-genetically matched precursor cells in mice. In animals with a form of Sandhoff disease, the approach helped them live longer and dramatically reduced behavioral and motor symptoms of the disease.
If the approach can be translated for use in humans, it could offer a glimmer of hope for the families of children with these rare diseases. Because it’s not necessary to use a patient’s own cells, the approach might one day offer the possibility of an “off the shelf” therapy that would be quicker and cheaper than bespoke genetic engineering for each case.
“Using a specific sequence of steps, we were able to achieve nearly 100% incorporation of genetically healthy cells in the brains of the mice while avoiding both rejection and graft-versus-host disease,” said professor of pathology Marius Wernig, MD, PhD. “This is vastly better than previous approaches. Furthermore, we were stunned at how well this therapy worked. The mice survived for the duration of the experiment, showed improved motor function and regained normal mouse behaviors like exploring open spaces. The difference between treated and control animals was dramatic.”
Wernig, a program leader in Stanford’s Institute for Stem Cell Biology and Regenerative Medicine, is the senior author of the research, to be published on Aug. 6 in Nature. Former postdoctoral scholar Marius Mader, MD, is the lead author of the study.
Devastating inherited diseases
Tay-Sachs, which affects about 1 in every 3,700 newborns of Ashkenazi Jewish descent, and Sandhoff, which is far less prevalent, belong to an inherited class of diseases called lysosomal storage disorders. Although rare, many are devastating, particularly those that primarily affect the brain. Affected infants often develop normally for the first weeks or months of life but rapidly regress as their neurons degenerate.
Babies born with these disorders have mutations in genes for enzymes critical to the function of lysosomes — cellular recycling compartments responsible for breaking down no longer needed proteins, carbohydrates and fat molecules called lipids into smaller building blocks for reuse. If lysosomes are unable to function, these molecules accumulate to dangerous levels in the cell.
But there is a mystery.
“Although the symptoms of these diseases are due to the degeneration of neurons, the levels of lysosomal enzymes in neighboring immune cells called microglia are sometimes a thousand-fold higher than in neurons,” said Wernig, the Dr. Salim and Mrs. Mary Shelby Faculty Scholar. So, why is it that the neurons are dying if the microglia are more severely affected?
One of the many functions of microglia is to engulf and break down dead cells or pathogens in the brain. “They are like professional cleaners,” Wernig said. “Therefore, they have a much greater need for these degradative enzymes than other cells.” The researchers wondered if restoring lysosomal enzymes to the microglia could somehow help the neurons.
Past attempts to correct the genetic deficiency in the microglia in lysosomal storage disorders involved a hematopoietic stem cell transplant (often referred to as a bone marrow transplant) to reboot the patient’s immune system, including the microglia in the brain. In this type of transplant, the person’s own immune system is first eliminated with drugs (a step called preconditioning), then genetically healthy immune cell precursors are introduced intravenously in the hope that they will establish themselves in the patient and begin making the missing protein.
But getting the healthy cells into the brain is difficult because the body tightly restricts access to the central nervous system. Other possible complications of a bodywide transplant include the elimination of donor cells by any remaining immune cells or, conversely, the life-threatening graft-versus-host disease.
Research from Wernig’s laboratory in 2022 showed it’s possible to achieve 90% engraftment of donor cells by wiping out the recipient animal’s immune system and treating them with a drug to kill off existing microglia, giving the donated hematopoietic stem cells a competitive growth advantage. But the technique still required toxic preconditioning and relied on genetically identical donor cells to prevent graft-versus-host disease.
“A hematopoietic stem cell transplant is a rough procedure to go through,” Wernig said. “It’s not something you want to do to your patients unless there’s no other option.”
A targeted transplant procedure
In the current study, Mader and Wernig tested whether they could develop a brain-specific transplant procedure that would avoid the toxic preconditioning and bodywide effects of a hematopoietic stem cell transplant. To that end, they coupled the microglia-depleting drug treatment with irradiation of the brain to create space for the new cells to occupy. They then injected microglia precursor cells — a more specialized subset of hematopoietic stem cells — from a non-genetically matched donor animal into the brain. Finally, they administered two drugs to block the activation of immune cells from elsewhere in the body that would otherwise kill the unmatched donated cells.
This delicate tango of steps resulted in efficient engraftment of the donated cells, which nestled into the brain and developed into microglia without migrating to the rest of the body or being attacked by the recipient animal’s immune system.
The cells’ engraftment was lasting: More than 85% of the microglial cells in the brain were derived from the donated cells eight months after transplantation. Untreated mice with a version of Sandhoff disease lived a median of 135 days and no animal lived beyond 155 days. In contrast, five of the animals treated with the brain-specific microglia transplantation therapy lived up to 250 days, when the experiment was terminated.
Although the long-lived treated mice eventually developed hind leg paralysis, they displayed normal exploration behaviors in a large open pen and greater muscle strength and coordination than control animals.
A closer investigation of the relationship between the donated microglia and their neuronal neighbors revealed something intriguing: The missing lysosomal enzyme now being made by the microglia could also be found in the animals’ native neurons. Although the reason is not yet known, Wernig and Mader suspect the microglia are packaging and secreting the enzyme into the space between cells, where it is imported into the neurons.
“This could be an important, unrecognized role for microglia: to supply lysosomal factors to the environment including neurons,” Wernig said.
The researchers are optimistic their approach could be translated to humans because each individual step — irradiation, administering drugs used to wipe out existing microglia and applying drugs to prevent immune attack of the donated cells — is already used to treat other conditions.
“We’ve solved three big problems with this study,” Wernig said. “We achieved efficient brain-restricted transplantation without systemic toxic preconditioning, we were able to use non-genetically matched cells that don’t require genetic engineering to make the missing lysosomal enzyme, and we avoided immune rejection and graft-versus-host disease. We’re very happy.”
The researchers also believe the therapy could be widely applicable.
“It’s possible that these lysosomal storage diseases are just an accelerated version of much more common neurodegenerative diseases like Alzheimer’s or Parkinson’s,” Wernig said. “If so, this therapy could be very relevant not just for a small subset of children, but for many, many more people.”
The study was funded by the California Institute for Regenerative Medicine, the German Research Foundation, the New York Stem Cell Foundation, the Robert J. Kleberg Jr. and Helen C. Kleberg Foundation, and the Wu Tsai Neurosciences Institute.
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