Caltech biologists have modified a harmless virus in such a way that it can successfully enter the adult mouse brain through the bloodstream and deliver genes to cells of the nervous system.

The blood-brain barrier allows the body to keep pathogens and potentially harmful chemicals circulating in the blood from entering the brain and spinal cord. The semi-permeable blockade, composed of tightly packed cells, is crucial for maintaining a controlled environment to allow the central nervous system to function properly. However, the barrier also makes it nearly impossible for many drugs and other molecules to be delivered to the brain via the bloodstream.

To sneak genes past the blood-brain barrier, the Caltech researchers used a new variant of a small, harmless virus called an adeno-associated virus (AAV). Over the past two decades, researchers have used various AAVs as vehicles to transport specific genes into the nuclei of cells; once there, the genes can be expressed, or translated, from DNA into proteins. In some applications, the AAVs carry functional copies of genes to replace mutated forms present in individuals with genetic diseases. In other applications, they are used to deliver genes that provide instructions for generating molecules such as antibodies or fluorescent proteins that help researchers study, identify, and track certain cells.

Largely because of the blood-brain barrier problem, scientists have had only limited success delivering AAVs and their genetic cargo to the central nervous system. In general, they have relied on surgical injections, which deliver high concentrations of the virus at the injection site but little to the outlying areas. Such injections are also quite invasive. "One has to drill a hole through skull, then pierce tissue with a needle to the injection site," explains the author. "The deeper the injection, the higher the risk of hemorrhage. With systemic injection, using the bloodstream, none of that damage happens, and the delivery is more uniform."

The researchers developed a high-throughput selection assay, CREATE (Cre REcombinase-based AAV Targeted Evolution), that allowed them to test millions of viruses in vivo simultaneously and to identify those that were best at entering the brain and delivering genes to a specific class of brain cells known as astrocytes.

They started with the AAV9 virus and modified a gene fragment that codes for a small loop on the surface of the capsid—the protein shell of the virus that envelops all of the virus' genetic material. Using a common amplification technique, known as polymerase chain reaction (PCR), they created millions of viral variants. Each variant carried within it the genetic instructions to produce more capsids like itself.

Then they used their novel selection process to determine which variants most effectively delivered genes to astrocytes in the brain. Importantly, the new process relies on strategically positioning the gene encoding the capsid variants on the DNA strand between two short sequences of DNA, known as lox sites. These sites are recognized by an enzyme called Cre recombinase, which binds to them and inverts the genetic sequence between them. By injecting the modified viruses into transgenic mice that only express Cre recombinase in astrocytes, the researchers knew that any sequences flagged by the lox site inversion had successfully transferred their genetic cargo to the target cell type—here, astrocytes.

After one week, the researchers isolated DNA from brain and spinal cord tissue, and amplified the flagged sequences, thereby recovering only the variants that had entered astrocytes.

Next, they took those sequences and inserted them back into the modified viral genome to create a new library that could be injected into the same type of transgenic mice. After only two such rounds of injection and amplification, a handful of variants emerged as those that were best at crossing the blood-brain barrier and entering astrocytes.

Through this selection process, the researchers identified a variant dubbed AAV-PHP.B as a top performer. To test AAV-PHP.B, the researchers used it to deliver a gene that codes for a protein that glows green, making it easy to visualize which cells were expressing it. They injected the AAV-PHP.B or AAV9 (as a control) into different adult mice and after three weeks used the amount of green fluorescence to assess the efficacy with which the viruses entered the brain, the spinal cord, and the retina.

"We could see that AAV-PHP.B was expressed throughout the adult central nervous system with high efficiency in most cell types," says the author. Indeed, compared to AAV9, AAV-PHP.B delivers genes to the brain and spinal cord at least 40 times more efficiently.  

"What provides most of AAV-PHP.B's benefit is its increased ability to get through the vasculature into the brain," says the author. "Once there, many AAVs, including AAV9 are quite good at delivering genes to neurons and glia."

The biologists conducted follow-up studies up to a year after the initial injections and found that the protein continued to be expressed efficiently. Such long-term expression is important for gene therapy studies in humans. They also showed that AAV-PHP.B is better than AAV9 at delivering genes to human neurons and glia.

http://www.caltech.edu/news/delivering-genes-across-blood-brain-barrier-49679