The go to gene-delivery vehicle, the adeno-associated virus (AAV), doesnt always go to where you would want it to go, particularly if the destinations you have in mind include the brain or the peripheral nervous system. Typically, viral vectors struggle to cross the bloodbrain barrier. Also, they tend to become dispersed across the body when they are tasked with delivering genetic cargo to far-flung neurons beyond the brain and spinal cord, such as those that sense pain and regulate heart rate, respiration, and digestion.

To help develop gene-delivery systems that can provide efficient transduction to neurons throughout the body, scientists based at the California Institute of Technology have introduced two AAV variants: one that efficiently ferries genetic cargo past the bloodbrain barrier and another that is efficiently picked up by peripheral neurons residing outside the brain and spinal cord.

Details appeared June 26 in the journal Nature Neuroscience, in an article entitled Engineered AAVs for Efficient Noninvasive Gene Delivery to the Central and Peripheral Nervous Systems. The vectors are able to reach their targets following a simple injection into the bloodstream. Also, the vectors are customizable and could potentially be used as part of a gene therapy to treat neurodegenerative disorders that affect the entire central nervous system, such as Huntington's disease, or to help map or modulate neuronal circuits and understand how they change during disease.

Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively, wrote the articles authors. In the adult mouse, intravenous administration of 1 1011 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 1012 vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons.

The work was led by Viviana Gradinaru, Ph.D., assistant professor of biology and biological engineering at Caltech. "We have now developed a new collection of viruses and tools to study the central and peripheral nervous systems," she said. "We are now able to get highly efficient brain-wide delivery with just a low-dose systemic injection, access neurons in difficult-to-reach regions, and precisely label cells with multiple fluorescent colors to study their shapes and connections."

The new vectors could help researchers study the activity and function of specific types of neurons within peripheral circuits using genetically encoded sensors and tools to modulate neuronal firing with light or designer drugs, respectively. The new vectors could also deliver genes that code for colorful fluorescent proteins, proteins that are useful in identifying and labeling cells.

The efficiency of these vectors facilitates robust cotransduction and stochastic, multicolor labeling for individual cell morphology studies, the articles authors noted. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity.

In the labeling process, multiple AAVseach carrying a distinct colorare mixed together and injected into the bloodstream. When they reach their target neurons, each neuron receives a unique combination of colors, thereby giving it a visually distinct hue that makes it easier for the researchers to distinguish its fine details from those of its neighbors. Furthermore, the team devised a technique to control the number of neurons labeledlabeling too many neurons makes it impossible to distinguish individual onesthat allows researchers to visualize individual neuron shapes and trace their connecting fibers through intact tissues using another technology that Dr. Gradinaru's laboratory has helped develop, known as tissue clearing.

"Usually, when researchers want a mouse or other animal model to express fluorescent proteins in certain cells, they need to develop genetically modified animals that can take months to years to make and characterize," said former graduate student and first author Ken Chan (Ph.D. '17). "Now with a single injection, we can label specific cells with a variety of colors within weeks after the injection."

"For our new systemic viral vectorsAAV PHP.S and AAV PHP.eBthere are many potential uses, from mapping circuits in the periphery and fast screening of gene regulatory elements to genome editing with powerful tools such as CRISPR/Cas9," asserted Dr. Gradinaru. "But perhaps the most exciting implication is that our tools, when paired with appropriate activity modulator genes, could enable noninvasive deep brain modulation for the treatment of neurological diseases such as Parkinson's disease."

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Gene Therapy Vectors Come to Grips with Nervous System, Root and Branch - Genetic Engineering & Biotechnology News