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The blood brain barrier (BBB) acts as a protective barrier for the central nervous system (CNS) by helping to maintain appropriate concentration gradients and acting as a defense shield against pathogens. In serving this vital role, the BBB denies most systemically administered molecules entry to the CNS. Therein lays the problem in treating CNS disorders, delivery. Therapeutic drug delivery is a common problem shared by both pharmacologists and gene therapists, but the field of viral gene delivery to the CNS may be on the verge of an exciting leap forward1, 2. This year, we reported on the ability of adeno-associated virus 9 (AAV9) to traverse the BBB when given as an intravenous (IV) infusion in both neonate and adult animals3, which was subsequently reported by a second independent group4. These were the first reports to demonstrate the ability to non-invasively deliver a product through the blood stream to target the brain and spinal cord, opening a plethora of basic research and therapeutic opportunities.
Administration of AAV9 to neonatal mice targeted neurons through out the entire brain. The reproducible transduction pattern suggests a distinct interaction between the virus and the tissue. A differential display of a receptor on a cell surface could explain the propensity of the virus for large neurons within the brain following IV infusion, but direct parenchymal injection targeted neurons within regions that were poorly transduced by systemic injection, such as those within the striatum. These results have led to numerous questions5, such as; What role does hemodynamics play in dictating the circulation of the virus? and, Are the neurons transduced simply a product of higher blood flow to them6?
Within spinal cord, the virus targeted lower motor neurons (LMNs) and dorsal root ganglia (DRG) cells. Approximately 60% of LMNs were transduced following AAV9 injection. To our knowledge, this is the highest percentage of LMN transduction with a single injection of any viral vector. Much of the surface area of these cells is outside the protection of the BBB, but the so-called “blood nerve barrier” should restrict access to the axons within the endoneurium, thus preventing them from being targeted by the virus. A retrograde transport dependent mechanism of transduction could explain the pattern within the spinal cord7. However, direct intramuscular injections showed little to no LMN or DRG transduction in adult animals3. The method of viral entry into these distinct cell populations remains a mystery, the unraveling of which could uncover means to increase efficiency of viral or non-viral drug delivery to the CNS.
Shockingly, the types of cells transduced changed from neuronal to glial when AAV9 was injected intravenously into adult animals. Transduction of astrocyte and microglial cells was observed throughout the entire CNS. The significance of this finding lies in the emergence of astrocytes and microglia as protagonists, and not just ancillary characters in neurodegenerative diseases8–10. Interestingly, AAV9 mediated glial transduction is only seen following IV infusion, while intraparenchymal injection results in the classic neuronal transduction pattern. Why is glial transduction route-dependent? There is precedent for regional distribution of surface receptors on astrocytes, rendering it possible for the viral receptor to be expressed only on the astrocytic endfeet that encase the blood vessels in the brain, thereby restricting access11. The mechanism of AAV9 entry into cells has not been fully resolved, therefore understanding the polarity of astrocytic transduction may aid in the determination of AAV9 receptors.
The predominant neuronal transduction seen in neonatal administration of AAV9 is contradictory to the glial transduction seen in adults, thus begging the obvious question as to what structural and molecular changes take place between neonates and adults. The simplest explanation is the presence or absence of astrocytes whose projections exist in contact with vascular endothelia. Astrogenesis in rodents occur predominantly within the first two postnatal weeks 12. Work in our lab is underway to understand the shift in AAV9 transduction. A similar puzzle, seemingly related to development, surrounds AAV9 transduction of LMNs with in the spinal cord. As previously mentioned, in neonates AAV9 transduces approximately 60% percent of LMN, while transduction in adults is ~5%. The precipitous drop in LMN infection remains to be explained. Besides developmental maturation, data in our lab suggests AAV9 LMN transduction is highly influenced by vector dose. Interestingly, volume is less a factor.
In summary, the finding that AAV9 can traverse the BBB in both adult and neonate animals avails a powerful tool to investigators interested in the CNS and its diseases. While our initial characterizations of AAV9 transduction largely focused on cells of the CNS involved in the motor and somatosensory systems, AAV9 targeting of the autonomic nervous system has not been evaluated. As in the 1844 childhood song: ‘To Grandmothers House We Go’, “Over the river and through the woods”, where will these vectors, such as AAV9 take us next?
The work described was supported by Award Number RC2NS069476, R01NS064492-01A1, R21EY018491-02 and R21NS064328-01 from the National Institute of Neurological Disorders And Stroke. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders And Stroke or the National Institutes of Health.