CREATE-ing tools that deliver genes past the blood-brain barrier

Your brain has a natural defense that protects it from infection and harm, it’s called the blood-brain barrier (BBB). The BBB is a selectively permeable layer of tightly packed cells that separates the blood in your circulatory system from your brain. Only certain nutrients, hormones, and molecules can pass through the BBB into the brain, while harmful chemicals and infection-causing bacteria are stopped at the border.

This ultimate defense barrier has its downsides though. It’s estimated that 98% of potential drugs that could treat brain diseases cannot pass through the BBB. Only some drug compounds that are very small in size or are fat-soluble can get through. Clearly, getting drugs and therapies past the BBB is a huge conundrum that remains to be solved.

Penetrating the Impenetrable

However, a CIRM-funded study published today in Nature Biotechnology has developed a delivery tool that can bypass the BBB and deliver genes into the brain. Scientists from Caltech and Stanford University used an innocuous virus called an adeno-associated virus (AAV) to transport genetic material through the BBB into brain cells.

Viral delivery is a common method to target and deliver genes or drugs to specific tissues or cells in the body. But with the brain and its impenetrable barrier, scientists are forced to surgically inject the virus into specific areas of the brain, which limits the areas of the brain that get treatment, not to mention the very invasive and potentially damaging nature of the surgery itself. For diseases that affect multiple areas in the brain, like Huntington’s and Alzheimer’s disease, direct injection methods are not likely to be effective. Thus, a virus that can slip past the BBB and reach all parts of the brain would be an idea tool for delivering drugs and therapies.

And that’s just what this new study accomplished. Scientists developed a method for generating modified AAVs that can be injected into the circulatory system of mice, pass through the BBB, and deliver genetic material into the brain.

They devised a viral selection assay called CREATE (which stands for Cre Recombinase-based AAV Targeted Evolution). Using CREATE, they tested millions of AAVs that all had slight differences in the genetic composition of their capsid, or the protein shell of the virus that protects the viruses’ genetic material. They tested these modified viruses in mice to see which ones were able to cross the BBB and deliver genes to support cells in the brain called astrocytes. For more details on how the science of CREATE works, you can read an eloquent summary in the Caltech press release.

A Virus that Makes Your Brain Glow Green

After optimizing their viral selection assay, the scientists were able to identify one AAV in particular, AAV-PHP.B, that was exceptionally good at getting past the BBB and targeting astrocytes in the mouse brain.

Lead author on the study, Ben Deverman, explained: “By figuring out a way to get genes across the blood-brain barrier, we are able to deliver them throughout the adult brain with high efficiency.”

They used AAV-PHP.B and AAV9 (which they knew could pass the BBB and infect brain cells) to transport a gene that codes for green fluorescent protein (GFP) into the mouse brain. After injecting mice with both viruses containing GFP, they saw that both viruses were able to make most of the cells in the brain glow green, confirming that they successfully delivered the GFP gene. When they compared the potency of AAV-PHP.B to the AAV9 virus, they saw that AAV-PHP.B was 40 times more efficient in delivering genes to the brain and spinal cord.

sing a new selection method, Caltech researchers have evolved the protein shell of a harmless virus, AAV9, so that it can more efficiently cross the blood brain barrier and deliver genes, such as the green fluorescent protein (GFP), to cells throughout the central nervous system. Here, GFP expression in naturally occurring AAV9 (left) can be seen distributed sparsely throughout the brain. The modified vector, AAV-PHP.B (right), provides more efficient GFP expression. Credit: Ben Deverman and the Gradinaru laboratory/Caltech - See more at:

Newly “CREATEd” AAV-PHP.B (right) is better at delivering the GFP gene to the brain than AAV9 (left). Credit: Ben Deverman.

“What provides most of AAV-PHP.B’s benefit is its increased ability to get through the vasculature into the brain,” said Ben Deverman. “Once there, many AAVs, including AAV9 are quite good at delivering genes to neurons and glia.”

Senior author on the study, Viviana Gradinaru at Caltech, elaborated: “We could see that AAV-PHP.B was expressed throughout the adult central nervous system with high efficiency in most cell types.”

Not only that, but using a neat technique called PARS CLARITY that Gradinaru developed in her lab, which makes tissues and organs transparent, the scientists were able to see the full reach of the AAV-PHP.B virus. They saw green cells in other organs and in the peripheral nerves, thus showing that AAV-PHP.B works in other parts of the body, not just the brain.

But just because AAV-PHP.B is effective in mice doesn’t mean it works well in humans. To address this question, the authors tested AAV-PHP.B in human neurons and astrocytes derived from human induced pluripotent stem cells (iPS cells). Sergiu Pasca, a collaborator from Stanford and author on the study, told the Stem Cellar:

Sergiu Pasca

Sergiu Pasca

“We have also tested the new AAV variant (AAV-PHP.B) in a human 3D cerebral cortex model developed from human iPS cells and have shown that it transduces human neurons and astrocytes more efficiently than does AAV9 demonstrating the potential for biomedical applications.”

An easier way to deliver genes across the BBB

This study provides a new way to cross the BBB and deliver genes and potential therapies that could treat a laundry list of degenerative brain diseases.

This is only the beginning for this new technology. According to the Caltech press release, the study’s authors have future plans for the AAV-PHP.B virus:

“The researchers hope to begin testing AAV-PHP.B’s ability to deliver potentially therapeutic genes in disease models. They are also working to further evolve the virus to make even better performing variants and to produce variants that target certain cell types with more specificity.”

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