In this study, we show for the first time that HDAd vectors delivered by intrathecal injection result in transduction and long-term transgene expression from neuroependymal and neuronal cells. Intravenous administration of the HDAd-CMV-LacZ vector indicated that the vector is not able to cross the blood–brain barrier in significant amounts, as shown by the minimal increase in β-galactoidase activity in the brain (), whereas other tissues (particularly the liver) had substantial activity (data not shown). Conversely, in intrathecal injected animals β-galactosidase activity was restricted to the brain.
At the microscopic level, intrathecal injection of HDAd vectors resulted primarily in transduction of ependymal cells lining the ventricles, which form the blood–CSF barrier surrounding both the brain and the spinal cord (). Interestingly, we have also shown that HDAd delivered by this route results in transduction of neurons located deep within the cerebral parenchyma at different time points post-vector injection. We reasoned that HDAd delivered by intrathecal injection infects the cells lining the ventricular system, which include a single layer of ependymal cells facing the lumen and the cells of the subventricular zone (SVZ) lying underneath the ependymal layer. It is well established that the SVZ of the lateral ventricles is a source of adult neuronal stem cells (NSCs). NSCs in the SVZ can differentiate into neurons in the olfactory bulbs and in the corpus callosum, as well as in fimbria and striatum oligodendrocytes. Although the ability of the ependyma to give rise to NSCs is still controversial (Ma et al.
), it is becoming clear that NSCs directly face the lateral ventricles through small apical processes (Mirzadeh et al.
), from where they likely come in contact with viral particles injected into the CSF space. Recently, also leptomeningeal compartment has been suggested to host a NSC niche (Bifari et al.
). Therefore, it is possible that HDAd delivered by intrathecal injection may allow transduction of NSCs that are subsequently found deep in the cerebral parenchyma. The detection of GFP+
cells in the cerebellum supports this hypothesis. In fact, Dcx expression in the adult brain was earlier considered to be restricted to the neuronal precursor phase of the neuronal lineage (Brown et al.
), while more recently Dcx+
neurosphere-forming cells were identified in the cerebellum of adult mouse (Walker et al.
). However whether Dcx expression represent just a stage before final commitment of stem cells to the neuronal lineage or whether this represents an entirely separate precursor population remains controversial.
Dcx is not expressed during gliogenesis or regenerative axonal growth (Couillard-Despres et al.
). For these reasons, it is unlikely that GFP+
cells result from the damage induced by the injection.
Intra-CSF injection (both intraventricular and lumbar puncture) of FGAd vectors expressing LacZ in nonhuman primates resulted in high transduction efficiency of leptomeningeal cells as shown by tissue staining at 72
hr post-injection (Driesse et al.
). Microscopic examination revealed transduction of arachnoid cells and to a lesser extent the cells of the pia mater. Ependymal and choroid plexus cells were also transduced (Driesse et al.
). Duration of transgene expression up to 3 months was observed in rhesus macaques injected with HDAd expressing GFP by lumbar puncture without signs of systemic or local toxicity or evidence of CNS-specific immune reaction (Butti et al.
; Terashima et al.
). However, in these studies transduction of neurons in the cerebral parenchyma has not been investigated. Our study also support that, similarly to intravascular delivery (Schnell et al.
; Brunetti-Pierri et al.
), intrathecal injection of HDAd results in a rapid dose-dependent acute inflammatory response (). This acute response is resolved by 48
hr post-injection and is likely the result of capsid-mediated activation of the innate immunity. Studies in large animal models are required to establish whether this response would be clinically acceptable.
An important finding of our study is that transgene expression is long term following intrathecal injection of HDAd vectors, at least in mice. Moreover, it indicates for the first time that this route of delivery results in transduction of neuronal cells. This finding is significant because targeting of neuronal cells is important for correction of several neurologic diseases and long-term expression is required for the treatment of genetic diseases affecting the CNS. In contrast to the limited vector distribution achieved by intracerebral injection, administration of HDAd vectors into the CSF has the potential advantage of widespread transduction along the cells lining the CSF space. Moreover, intrathecal injection is a far less invasive procedure than intracerebral injection and therefore is attractive for clinical applications.
The transduced ependymal cells could be potentially used to secrete therapeutic proteins into the CSF. As previously shown this route of administration resulted in the production of significant amounts of bioactive proteins which can be exploited for multiple therapeutic purposes (Betz et al.
; Furlan et al.
). Transduced cells could secrete the therapeutic protein via the CSF for cross-correction of nontransduced cells. Thus, this approach may be particularly attractive for lysosomal storage diseases in which cross-correction is mediated by the mannose-6-phosphate receptor. It remains to be seen whether intrathecal administration of an HDAd vector is equally effective in larger mammals where greater diffusion distances may limit effective distribution of the vector and/or its therapeutic product.