The findings presented in this study demonstrate a unique capacity for AAV9-vectors to cross the BBB in varying degrees given the timepoint of gene administration. The delivery of AAV9-GFP in the neonate resulted in widespread neuronal targeting with an apparent tropism towards motor neurons residing within the spinal cord. Within numerous regions of the brain, we found substantial transduction of neurons. Interestingly, based upon our data, GABAergic interneurons were not highly transduced by AAV9 using a vascular delivery approach, yet pyramidal and Purkinje neurons were highly transduced. These transduced cells have long projection axons, unlike interneurons. Our results may postulate that the specific neuroanatomy, such as neuronal morphology or vascular density may determine the capacity of which neurons are transduced by AAV9 in this injection paradigm. These results justify further study which may provide insight into the mechanisms for viral gene transfer of AAV9-based vectors to specific neuronal populations.
The scarcity of LMN and DRG transduction seen in the adult paradigm suggests there is a developmental period in which access by circulating virus to these cell populations becomes restricted. Assuming a dependence on retrograde transport for DRG and LMN transduction following intravenous injection, Schwann cell or synapse maturation may be an important determinant of successful AAV9 LMN and DRG transduction. Previously, we reported retrograde transport by AAV vectors20, 21
and recently some serotypes have demonstrated an increased capability for retrograde transport22
. However, we did not detect obvious retrograde transport from AAV9 vectors following intramuscular injection in adult treated animals.
The predominant astrocyte transduction in adults suggests that AAV9 escapes brain vasculature in a similar manner as skeletal and cardiac muscle vasculature. The ability for AAV9 to penetrate or bypass endothelium is of interest. Normally, tight junctions severely restrict BBB penetration of molecules, such as viruses. AAV9 may be utilizing transport proteins, receptor mediated transcytosis, adsorptive transcytosis or other mechanisms to gain access across the BBB. Once free of the vasculature, our data suggests that AAV9 infects the astrocytic-perivascular-endfeet that surround capillary endothelial cells23
. Our results highlight the interesting aspect that astrocytes are targeted only when the virus is exposed to the astrocytic perivascular-endfeet. Given that astrocytes retain some features of apical-basal polarity from development, certain receptors or channels may be accessible via this vascular entry route compared to direct intraparencymal injections24
. The precise mechanism of bypassing endothelial cells leading to astrocytic transduction will require further study.
In summary, our results demonstrate the unique capacity of AAV9 to efficiently target cells within the CNS, and in particular, widespread neuronal and motor neuron transduction in the neonate, and extensive astrocyte transduction in the adult following intravenous delivery. A simple intravenous injection of AAV9 as we describe here may be clinically relevant for both SMA and ALS. In the context of SMA, data suggests that increased expression of survival motor neuron (SMN
) gene in LMNs may hold therapeutic benefit3, 25
. The importance of the results presented here is that with a single injection we may be able to effectively restore SMN
expression levels in LMNs. Additionally, given the robust neuronal populations transduced throughout the CNS in neonatal animals, this approach may also allow for rapid, relatively inexpensive generation of chimeric animals for gene overexpression, or gene knock-down26
. Additionally, constructing AAV9 based vectors with neuronal or astrocyte specific promoters may allow further specificity, given that AAV9 targets multiple non-neuronal tissues following intravenous delivery11, 13
. Our results also demonstrated efficient targeting of astrocytes in adult-treated animals, and this finding may be relevant for treating ALS, where the non-cell autonomous nature of disease progression has recently been discovered, and astrocytes have been specifically linked to disease progression27
. The ability to target astrocytes for producing trophic factors, or to circumvent aberrant glial activity may be beneficial for treating ALS28
. Noteworthy, AAV9 vascular delivery in the adult does not achieve widespread direct neuronal targeting, limiting this approach for diseases such as Huntington's where multiple structures and brain regions likely require neuronal targeting to suppress mutant Huntington29, 30
. However, the ability to generate viruses that are more efficient and reach neurons in the adult by vascular delivery may be possible with newly generated techniques in viral evolution based on AAV9 capsids31–33
. In sum, our results highlight a relatively non-invasive method to efficiently deliver genes to the CNS and may be useful in basic and clinical neurology studies.