‘Therapeutic EVs’ in a Nasal Spray Could One Day Treat Alzheimer’s Disease
By Deborah Borfitz
December 5, 2024 | It sounds too good to be true: a nasal spray that delays Alzheimer’s-related behavioral and cognitive problems a decade or more after initial diagnosis. But promising first steps in that direction have already been taken using “therapeutic EVs” (extracellular vesicles) restricting the activation of inflammatory cytokines in the brain of mice, according to Ashok K. Shetty, Ph.D., associate director of the Institute for Regenerative Medicine at Texas A&M University.
EVs have received significant attention in the therapeutic field because, in fulfilling their function in intercellular communication, they naturally carry multiple types of bioactive molecules such as proteins and nucleic acids that can be delivered to specific target cells to reduce neuroinflammation, he explains. While other labs are testing them in animal models of Alzheimer’s disease, his group has conducted one of the first studies showing that intranasal administration of neural stem cell-derived EVs can get into the brain, interact with the neural cells, and postpone mental decline. That study was published recently in the Journal of Extracellular Vesicles (DOI: 10.1002/jev2.12519).
It is a different starting point than with liquid biopsies that track neuroinflammation in the brain by isolating and characterizing microglial, astrocyte, and neuron EVs in circulating blood and studying their cargo. Here, researchers begin with the human neural stem cells derived from pluripotent stem cells and use molecular assays to characterize the multiple microRNAs and proteins that fill the shed EVs, which could restrain various neuropathological changes in animal models of Alzheimer’s disease, says Shetty.
In doing so, they have discovered that the intake of the intranasally administered EVs into microglia dramatically changes the gene expression of the cells to confer a neuroprotective effect while not interfering with the ability of these resident immune cells to continue clearing the disease-related amyloid beta and tau buildup, he shares. The same phenomenon was witnessed with astrocytes, which also contribute to neuroinflammation in Alzheimer’s disease.
The non-invasive method for targeting EVs to the brain by administering them into the nasal cavity has separately been used by Shetty and his colleagues in models of traumatic brain injury (TBI) and other neurological diseases. He has a patent application pending on the intranasal application of neural stem cell-derived EVs covering all neurological and neurodegenerative disorders displaying neuroinflammation—a list that includes TBI and Alzheimer’s as well as stroke and Parkinson’s disease.
Exploring the Secretome
Shetty has been working in the stem cell field for several decades. His previous preclinical studies have demonstrated the efficacy of cell therapy for temporal lobe epilepsy using human pluripotent stem cell-derived interneurons. This concept is now being pursued in clinical trials by San Francisco-based Neurona Therapeutics, which developed a treatment in parallel with published studies from Shetty’s laboratory on interneuron cell therapy (npj Regenerative Medicine, DOI: 10.1038/s41536-022-00234-7). But Alzheimer’s disease affects multiple regions of the brain, making the transplantation of cells impractical, he says.
For this reason, he and his team began looking at the stem cell secretome—the collection of molecules, including proteins, growth factors, cytokines, and EVs secreted by stem cells into their surrounding environment—in search of those having therapeutic properties that might instead be leveraged. When administered intranasally, the tiny, stem cell-derived EVs can easily get into every region of the brain within 60 minutes, says Shetty, highlighting the beauty of the approach. This eliminates the need to inject stem cells into the brain, which comes with bleeding and infection risks and can potentially cause tumor formation or get stuck within capillaries and cause thrombosis—neither of which are issues with therapeutic EVs.
For the study in Alzheimer’s mice, researchers directly placed EVs into the nostrils, he explains. “This can be developed into a nasal spray for human use in the future.”
Intranasal administration gives the EVs easy access to the subarachnoid space, which is filled with cerebrospinal fluid (CSF) that protects and cushions the brain and spinal cord. “Once they get in the subarachnoid space, they move with CSF flow... into the perivascular space and then into the interstitial space,” he says. It is here—within this network of tiny, fluid-filled gaps between neurons and glial cells—that the therapeutic EVs get taken up by various cell types found in the brain.
Importantly, activated microglia and reactive astrocytes in the Alzheimer’s brain interact with EVs. The initial activation of these glial cells is considered beneficial because they can efficiently remove harmful amyloid beta and tau from the brain, says Shetty. But when super-activated as Alzheimer’s disease progresses, they start to release multiple inflammatory cytokines. Increased concentrations of pro-inflammatory cytokines in the brain can lead to loss of synapses, the connections between neurons, and eventually to neurodegeneration.
In the latest study, Shetty and his colleagues succeeded in demonstrating how the interaction of EVs with both activated microglia and reactive astrocytes restrained neuroinflammatory signaling cascades. Notably, single-cell RNA sequencing showed that the expression of multiple genes involved in the super-activation of these cells was altered by the therapeutic EVs so “they became less active, but they didn’t stop doing their job” of clearing the proteins involved in Alzheimer’s disease, Shetty says.
The result was maintenance of better cognitive and mood function in the animals three months later, as measured by a battery of neuro-behavioral and -cognitive tests, relative to the untreated animals who displayed dysfunctions, he adds. Three months in mouse years is roughly equivalent to 10 human years.
Road to the Clinic
Intranasally administered EVs also interact with neurons in the brain, says Shetty, which is the subject of an ongoing investigation in Alzheimer’s mice. Cell culture studies in his laboratory have suggested that human neurons taking up the neural stem cell-derived EVs develop resistance to amyloid beta-induced tau phosphorylation and neurodegeneration.
For the preclinical studies, the therapeutic EVs being used are all naturally released into the secretome by neural stem cells, he notes. “The next step would be to further enhance therapeutic efficacy by loading certain therapeutic compounds into these EVs” for delivery to the brain.
The research focus currently is on intervening in early stages of Alzheimer’s disease and seeing how long better cognitive and mood function can be maintained, says Shetty. “We are continuing those studies and looking at animals over more extended timelines after the initial administration.” Simultaneously, investigators are intervening in later stages of the disease “to see whether we can reverse some of the cognitive changes that have already occurred.”
Funding support is coming from the National Institute for Aging through an RO1 grant. Clinical translation will take several years and require multiple steps, the first being generation of clinical-grade EVs grown in a facility that adheres to Good Laboratory Practices using compatible reagents for eventual testing in patients through clinical trials, he says. Toxicity studies and testing in larger animal models will also be needed.
One clinical possibility is that therapeutic EVs could be administered intranasally to people newly diagnosed with Alzheimer’s disease to delay the development of cognitive and mood problems, says Shetty. The research team is also conducting experiments in aged animals to learn if the treatment might prevent people with mild cognitive impairment from going on to develop Alzheimer’s disease.