Novel subventricular zone early progenitor cell-specific adenovirus for in vivo therapy of central nervous system disorders reinforces brain stem cell heterogeneity.

Neural stem/progenitor cells (NSPCs) have the potential to self-renew and to generate all neural lineages as well as to repopulate damaged areas in the brain. Our previous targeting strategies have indicated precursor cell heterogeneity between different brain regions that warrants the development of NSPC-specific delivery vehicles. Here, we demonstrate a target-specific adenoviral vector system for the in vivo manipulation of progenitor cells in the subventricular zone of the adult mouse brain. For this purpose, we identified a series of peptide ligands via phage display. The peptide with the highest affinity, SNQLPQQ, was expressed in conjunction with a bispecific adaptor molecule. To verify the targeting potential of the specific peptide, green fluorescent protein-expressing Ad vectors were coupled with the adaptor molecule and injected into the subventricular region of adult mice by stereotaxic surgery. An efficient and selective transduction of NSPCs in the SVZ was achieved, whereas hippocampal NSPCs were negative. Our results offer an expeditious and simple tool to produce retargeted viral vectors for a specific and direct in vivo manipulation of these progenitor cells. This powerful technique provides an opportunity to develop innovative strategies and express therapeutic genes in specific types of neural progenitor cells to allow success in treatment of brain disorders.

Impact of Toll-like receptor 2 deficiency on survival and neurological function after cardiac arrest: a murine model of cardiopulmonary resuscitation.

BACKGROUND: Cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR) is associated with poor survival rate and neurofunctional outcome. Toll-like receptor 2 (TLR2) plays an important role in conditions of sterile inflammation such as reperfusion injury. Recent data demonstrated beneficial effects of the administration of TLR2-blocking antibodies in ischemia/reperfusion injury. In this study we investigated the role of TLR2 for survival and neurofunctional outcome after CA/CPR in mice. METHODS: Female TLR2-deficient (TLR2(-/-)) and wild type (WT) mice were subjected to CA for eight min induced by intravenous injection of potassium chloride and CPR by external chest compression. Upon the beginning of CPR, n = 15 WT mice received 5 µg/g T2.5 TLR2 inhibiting antibody intravenously while n = 30 TLR2(-/-) and n = 31 WT controls were subjected to injection of normal saline. Survival and neurological outcome were evaluated during a 28-day follow up period. Basic neurological function, balance, coordination and overall motor function as well as spatial learning and memory were investigated, respectively. In a separate set of experiments, six mice per group were analysed for cytokine and corticosterone serum levels eight hours after CA/CPR. RESULTS: TLR2 deficiency and treatment with a TLR2 blocking antibody were associated with increased survival (77% and 80% vs. 51% of WT control; both P < 0.05). Neurofunctional performance was less compromised in TLR2(-/-) and antibody treated mice. Compared to WT and antibody treated mice, TLR2(-/-) mice exhibited reduced IL-6 (both P < 0.05) but not IL-1ß levels and increased corticosterone plasma concentrations (both P < 0.05). CONCLUSION: Deficiency or functional blockade of TLR2 is associated with increased survival and improved neurofunctional outcome in a mouse model of CA/CPR. Thus, TLR2 inhibition could provide a novel therapeutic approach for reducing mortality and morbidity after cardiac arrest and cardiopulmonary resuscitation.

Targeting of neural stem cells in the hippocampus of adult rats by custom-made Ad vectors.

Adult hippocampal neural stem cells (NSC) are an intriguing source for cell replacement or could serve as delivery vehicles for therapeutic genes. We recently reported selective transduction of adult mouse NSC in the DG by in vivo injection of GFP encoding adenoviral (Ad) vectors engineered to bind NSC-specific peptides. Here, we investigated the specificity of these peptide-tagged vectors in the adult rat DG, and whether they can be used to follow differentiation of infected cells over time. The virus-containing solution was injected into the DG by stereotaxic surgery. Specific transduction of NSC was demonstrated by the radial glia-like morphology of GFP-expressing type-1 cells and co-labeling with nestin or glial fibrillary acidic protein. Three days post-injection more than 82% of GFP-containing cells were nestin-immunoreactive, as revealed by unbiased stereology and no GFP-expressing neurons were observed. However, 30 days after injection, the amount of GFP and nestin-containing cells declined (56%), whereas now neurons that contained NeuN or possessed the typical granular nerve cell morphology expressed GFP, indicating that they were derived from initially transduced NSC. Importantly, still more than 20% of nestin-immunoreactive NSC was found to be GFP-positive 90 days after infection, but unfortunately at this time point no GFP-containing neurons were detectable. Our results demonstrate that Ad vectors tagged with NSC-specific ligands can be used to target type-1 NSC, the low-proliferating cell population, in the rat hippocampus. They are a valuable tool to monitor the differentiation of their descendants, at least over short time periods.

Selective targeting of adenoviral vectors to neural precursor cells in the hippocampus of adult mice: new prospects for in situ gene therapy.

The adult brain contains neural precursor cells (NPC) that are attracted to brain lesions, such as areas of neurodegeneration, ischemia, and cancer. This suggests that NPC engineered to promote lineage-specific differentiation or to express therapeutic genes might become a valuable tool for restorative cell therapy and for targeting therapeutic genes to diseased brain regions. Here we report the identification of NPC-specific ligands from phage display peptide libraries and show their potential to selectively direct adenovirus-mediated gene transfer to NPC in adult mice. Identified peptides mediated specific virus binding and internalization to cultured neurospheres. Importantly, peptide-mediated adenoviral vector infection was restricted to precursor cells in the hippocampal dentate gyrus of pNestin-green fluorescent protein transgenic or C57BL/6 mice. Our approach represents a novel method for specific manipulation of NPC in the adult brain and may have major implications for the use of precursor cells as therapeutic delivery vehicles in the central nervous system.