Successful delivery of genes through the blood-brain barrier

Written by Alice Weatherston

Potentially groundbreaking research, published recently in Nature Biotechnology, has successfully modified an adeno-associated virus (AAV) to deliver genes across the blood-brain barrier and into a mouse brain. The research, carried out at Caltech (CA, USA), provides hope for the delivery of novel therapeutics into the brain.
“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,” explained lead author Ben Deverman.

To date, success in delivering AAV’s and associated genes to the brain and central nervous system has been limited, requiring invasive surgical injections into the skull in many cases. These injections also only deliver the virus to localized areas, reducing their efficacy for disorders such as neurodegenerative diseases that affect large areas of the brain and psychological disorders which involve many interacting neuronal networks.

Vivian Gradinaru, senior author of the study, set out to build on work published in 2009 by Brian Kaspar (Ohio State University; OH, USA) and his team, which revealed that the AAV strain termed AAV9 was capable of entering the brains of neonatal or infant mice, but not adults, following injection into the bloodstream.

The research team developed a high-throughput selection assay (CREATE) that enabled them to test millions of viruses simultaneously in vivo in order to identify candidate viruses that would be capable of acting as a successful gene vector. Starting with the AAV9 virus, viral variants were produced carrying genetic coding for the development of capsids. This gene encoding was placed at a specific lox site recognized by the enzyme termed Cre recombinase. The modified viruses were then injected directly into transgenic mice only expressing Cre recombinase in astrocytes, enabling sequences at the lox site to be flagged specifically and therefore identification of successfully transferred genetic code into the astrocytes.

One week after injection the research team analyzed isolated DNA from brain and spinal cord tissue, recovered the variant that had successfully entered the astrocytes and reinserted them back into the viral genome and into transgenic mice. After two rounds this process revealed a variant termed AAV-PHP.B as the most efficacious.

In further tests AAV-PHP.B was utilized to deliver a gene encoding a fluorescent protein into adult mice – AAV9 was also inserted into different adult mice as a control. After three weeks, analysis of fluorescence within the brain, spinal cord and retina indicated that AAV-PHP.B delivers genes to these areas at least 40 times more efficiently than AAV9. “We could see that AAV-PHP.B was expressed throughout the adult central nervous system with high efficiency in most cell types,” explained Gradinaru.

Follow-up studies up to a year after initial injections also indicated that the fluorescent protein delivered by AAV-PHP.B continued to be well expressed.

“What provides most of AAV-PHP.B’s benefit is its increased ability to get through the vasculature into the brain,” commented Deverman.

The researchers now hope to commence testing of AAV-PHP.B’s ability to deliver potentially therapeutic genes in disease models and are working to evolve the virus further to generate even better performing and more targeted variants.

“The CREATE system gave us a good hit early on, but we are excited about the future potential of using this approach to generate viruses that have very good cell-type specificity in different organisms, especially the less genetically tractable ones,” remarked Gradinaru. “This is just the first step. We can take these tools and concepts in many exciting directions to further enhance this work.”

Source: Caltech press release